Non-recombinant Saccharomyces strains that grow on xylose

ABSTRACT

The present invention relates to methods for producing  Saccharomyces  strains that are capable of growth on xylose as a sole carbon source at a desired growth rate (such as at least one generation per 48 hours), strains made by such methods, and  Saccharomyces  strains that grow at a growth rate of at least one generation per 48 hours using xylose as a sole carbon source for growth made by non-recombinant methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional (and claims the benefit of priorityunder 35 U.S.C. §121) of U.S. Application Ser. No. 11/570,329, filedDec. 8, 2006, which claims priority from PCT Application Ser. No.PCT/AU2005/000824, filed Jun. 8, 2005, which claims priority fromAustralian Application Ser. No. 2004903141, filed Jun. 8, 2004. Thecontents of these applications are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The invention relates to methods for producing non-recombinant strainsof Saccharomyces, strains of Saccharomyces, and uses thereof.

BACKGROUND OF THE INVENTION

All references, including any patents or patent applications, cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. The discussion of thereferences states what their authors assert, and the applicants reservethe right to challenge the accuracy and pertinency of the citeddocuments. It will be clearly understood that, although a number ofprior art publications are referred to herein, this reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art, in Australia or in any othercountry.

One of the most important economic groups of yeasts are of the genusSaccharomyces, strains of which are employed in the brewing, baking,winemaking, distilling and various other yeast-dependent industries.Species of Saccharomyces are as defined phylogenetically by Kurtzman(2003) FEMS Yeast Research 3:417-432, and include S. cerevisiae, S.paradoxus, S. mikatae, S. cariocanus, S. kudriavzevii, S. pastorianusand S. bayanus.

Saccharomyces spp. are some of the most effective microorganisms forconverting sugars such as glucose, fructose, sucrose and maltose tobiomass, and for fermenting these sugars to ethanol. As a consequence,Saccharomyces spp., and in particular Saccharomyces cerevisiae, is oneof the most widely used microorganisms in industrial processes. Forexample, in the beer brewing, distilling and wine industries,Saccharomyces are used to ferment sugars such as glucose, fructose,sucrose and/or maltose into ethanol. In the fuel ethanol industry,Saccharomyces cerevisiae strains are chosen for their ability to rapidlyconvert high concentrations of sugars such as glucose, fructose, sucroseand maltose into high amounts of ethanol. In the baking industry,Saccharomyces cerevisiae strains are used primarily for their ability toproduce carbon dioxide from sugars such as glucose, fructose, sucrose,and/or maltose in order to leaven bread. Other applications ofSaccharomyces cerevisiae include production of yeast extracts and otherflavour and aroma products, sources of enzymes such as invertase,production of various biochemicals, intermediates, proteins, aminoacids, ribonucleic acid and nucleotides co-factors and vitamins.

Millions of tonnes of yeast are grown each year in industrial processes.Therefore, the ability of Saccharomyces to grow on abundant andrenewable carbon sources is important in terms of economic production ofyeast biomass for industrial purposes and for economic production ofbyproducts from yeast metabolism. In particular the ability ofSaccharomyces to grow on waste byproducts of other industrial processesis of environmental and economic value. Thus, for example, baker's yeastbiomass is often produced by growing yeast on molasses which is a wasteproduct of the sugar production process, or on glucose- and maltose-richsyrups derived from starch hydrolysis industry.

SUMMARY OF THE INVENTION

The invention provides methods of producing a Saccharomyces strain thatis capable of growing at a desired growth rate (such as at least onegeneration per 48 hours), using xylose as a sole carbon source, andstrains of Saccharomyces that grow on xylose under these growth rates,and the uses thereof.

Xylose is an example of a naturally occurring, abundant and renewablesugar available from plant biomass that Saccharomyces was previouslyconsidered not able to use. See for example, Barnett et al. ‘YeastsCharacteristics and Identification’ 2nd edition (1990), CambridgeUniversity Press, which states that yeast of the species Saccharomycescerevisiae are not capable of utilizing xylose as a sole carbon sourcefor growth. Xylose represents a major potential source for growingyeasts and for manufacturing products, including ethanol, from yeasts.The use of xylose as a sugar source for yeasts would provide majoreconomic and environmental advantages. For example, production of fuelethanol from xylose-rich materials would be a major source of renewableenergy.

In a first aspect, the invention provides a method of producing aSaccharomyces strain that is capable of growing at a desired growth rateusing xylose as a sole carbon source for growth, comprising:

-   -   (a) providing a population of genetically diverse        non-recombinant yeast cells of Saccharomyces;    -   (b) culturing the yeast cells under conditions which permits        combining of DNA between yeast cells;    -   (c) screening or selecting the yeast cells for yeast cells which        have increased growth rate using xylose as a sole carbon source        for growth;    -   (d) isolating one or more yeast cells which have the desired        growth rate.

Typically, the yeast cells are cultured under conditions which permitcombining of DNA between pairs of yeast cells.

Typically, the desired growth rate is an increase in growth raterelative to that of yeast cells of Saccharomyces strains to which themethod has not been applied. For example, Saccharomyces strains such asstrain NL67. More typically, the desired growth rate is at least onegeneration per 48 hours, more typically at least one generation per 24hours.

Typically, the yeast cells are screened or selected for yeast cellswhich have the desired growth rate by incubating the yeast cells in oron xylose-containing medium. Typically, the xylose-containing medium isxylose-containing culture medium. The culture medium may contain xyloseas the only carbon source, or xylose may be one of a plurality of carbonsources. Typically, the xylose is the only carbon source in thexylose-containing medium. Typically, the xylose-containing culturemedium is xylose minimal mineral media. The yeast cells are typicallyincubated on or in the xylose-containing medium for sufficient time topermit yeast cells having the desired growth rate using xylose as acarbon source to grow.

In one embodiment, the screened or selected yeast cells form thepopulation of genetically diverse non-recombinant yeast cells, and steps(b) and (C) are repeated until yeast cells having the desired growthrate are obtained.

In one embodiment, step (b) is performed prior to step (c). In anotherembodiment, step (c) is performed before step (b). In yet anotherembodiment, steps (b) and (c) are performed simultaneously.

In a second aspect, the invention provides a method of producing aSaccharomyces strain in culture that is capable of a desired growth rateusing xylose as a sole carbon source for growth, comprising:

-   -   (a) providing a population of genetically diverse        non-recombinant yeast cells of Saccharomyces;    -   (b) culturing the yeast cells under conditions which permits        combining of DNA between yeast cells;    -   (c) screening or selecting the yeast cells by incubating the        yeast cells on or in a xylose-containing medium;    -   (d) repeating steps (b) and (c) with the screened or selected        cells forming the population of genetically diverse        non-recombinant yeast cells of Saccharomyces, until one or more        yeast cells have acquired the ability to grow at a desired        growth rate using xylose as a sole carbon source for growth.

Typically, the method comprises the further step (e) of isolating one ormore yeast cells having the desired growth rate. In one embodiment, theone or more yeast cells that is isolated is a single strain ofSaccharomyces. In another embodiment, the one or more yeast cells thatis isolated is a population of genetically diverse yeast cells.

In one embodiment, the yeast cells are selected in step (c). The yeastcells may be selected in step (c) by incubating the yeast cells on or inxylose-containing medium for sufficient time to permit yeast cellscapable of growth on xylose as a sole carbon source to grow using xyloseas the carbon source, and thereafter collecting the yeast cells thatgrow. The yeast cells may be selected in step (c) by incubating theyeast cells on or in xylose-containing medium for sufficient time topermit yeast cells having an increased growth rate using xylose as asole carbon source to grow using xylose as the carbon source, andthereafter collecting the yeast cells that grow.

In another embodiment, the yeast cells are screened in step (c). Theyeast cells may be screened in step (c) by incubating the yeast cells onor in xylose-containing medium for sufficient time to permit yeast cellshaving an increased growth rate using xylose as a sole carbon source togrow using xylose as the carbon source, and thereafter collecting theyeast cells that have an increased growth rate using the xylose as acarbon source.

It is also envisaged that in a portion of the repeats of steps (b) and(c), the yeast cells will be selected, and in a portion of the repeatsof steps (b) and (c), the yeast cells will be screened.

Steps (b) and (c) may be repeated any number of times that is sufficientto obtain a yeast strain that is capable of growth at the desired rateusing xylose as a sole carbon source for growth. It will be appreciatedby persons skilled in the art that the number of times that steps (b)and (c) will be repeated will depend upon the medium which is used, thestarting yeast strains, the culturing conditions, etc. In oneembodiment, steps (b) and (c) are repeated at least once, typically atleast 2 times, more typically at least 5 times, even more typically atleast 10 times, still more typically at least 20 times, suitably atleast 30 times.

The yeast cells may be screened or selected on solid or in liquidxylose-containing medium. In one embodiment, the cells are screened orselected on solid xylose-containing medium. In another embodiment, thecells are screened Or selected in liquid xylose-containing medium. Inyet another embodiment, the cells are screened or selected on solidxylose-containing medium, followed by screening or selection in liquidxylose-containing medium. For example, the yeast cells may be screenedor selected for a plurality of times by repeating steps (b) and (c)using solid xylose-containing medium, followed by screening or selectionfor a plurality of times by repeating steps (b) and (c) using liquidxylose-containing medium.

The solid xylose-containing medium may be any solid medium that containsxylose as a carbon source, and which provides a selective advantage to astrain that is capable of utilising xylose as a sole carbon source. Forexample, the solid xylose-containing medium may be a complex solidmedium in which xylose is one of a plurality of carbon sources, or thesolid xylose-containing medium may be a minimal solid medium in whichxylose is the sole carbon source. Typically, the solid xylose-containingmedium will be solid minimal medium containing xylose as a sole carbonsource.

The liquid xylose-containing medium may be any liquid medium thatcontains xylose as a carbon source. For example, the liquidxylose-containing medium may be a complex liquid medium in which xyloseis one of a plurality of carbon sources, or the liquid xylose-containingmedium may be a minimal liquid medium in which xylose is the sole carbonsource. Typically, the liquid xylose-containing medium will be liquidminimal medium containing xylose as a sole carbon source. Typically, theliquid minimal medium is minimal mineral medium containing xylose as asole carbon source.

The desired growth rate is typically at least one generation per 48hours. The desired growth rate may be greater than one generation per 24hours. The desired growth rate may be greater than one generation per 12hours. The desired growth rate may be greater than one generation per 10hours. The desired growth rate may be greater than one generation per 8hours. The desired growth rate may be greater than one generation per 4hours. The desired growth rate may be greater than one generation per 2hours.

The Saccharomyces strain that is produced may be a strain from anyspecies of the genus Saccharomyces that is capable of growth at adesired growth rate using xylose as a sole carbon source for growth. Itwill be appreciated by those skilled in the art that the strain that isproduced will depend on the species which form the genetically diversepopulation of non-recombinant yeast cells of Saccharomyces. Examples ofsuitable species of yeast include S. cerevisiae, S. paradoxus, S.mikatae, S. cariocanus, S. kudriavzevii, S. pastorianus and S. bayanus.Typically the strain is of the species Saccharomyces cerevisiae.Typically the strain will be capable of mating with Saccharomyces of thesame species. Typically the strain will be capable of mating withSaccharomyces cerevisiae.

The yeast may be cultured under any conditions which permit combining ofDNA between yeast cells provided the combining of DNA is not byrecombinant methods. The yeast cells may be cultured under conditionswhich permit combining of DNA between yeast cells by, for example,mating or cytoduction, or any other methods known in the art forcombining of DNA of yeast cells, other than recombinant methods. In oneembodiment, the yeast cells are cultured under conditions which permitmating of the yeast cells. Typically, mating of yeast comprisessporulating the yeast to produce spores, germinating the spores, andmating the germinated spores. Methods for mating yeast are known tothose skilled in the art and are described in, for example, Fowell(1969) “Sporulation and hybridization of yeast”, in, The Yeasts (A HRose and J S Harrison, eds.), Academic Press; European Patent EP 0 511108 B; Attfield and Bell (2003) “Genetics and classical geneticmanipulations of industrial yeasts” in, Topics in Current Genetics, Vol2. Functional Genetics of Industrial Yeasts (J. H. de Winde, ed.),Springer-Verlag Berlin Heidelberg or combinations thereof.

The population of genetically diverse non-recombinant yeast cells may benaturally-occurring isolates of Saccharomyces from any source,spontaneously mutated isolates of Saccharomyces, or may be obtained byexposing one or more Saccharomyces strains to a mutagen. The populationof genetically diverse non-recombinant yeast cells may be strains from asingle species, or may be strains from a plurality of species. Speciessuitable for use as the genetically diverse population ofnon-recombinant yeast cells includes S. cerevisiae, S. paradoxus, S.mikatae, S. cariocanus, S. kudriavzevii, S. pastorianus and S. bayanus.Typically the strains are of the species Saccharomyces cerevisiae.Typically the strains are capable of mating with Saccharomyces of thesame species. Typically the strains are capable of mating withSaccharomyces cerevisiae.

It will be understood by persons skilled in the art that in addition tothe population of genetically divergent non-recombinant yeast cells,recombinant yeast cells may be included in steps (b) and (c) as aseparate population.

In a third aspect, the invention provides a method of generating aderivative of a Saccharomyces strain with an increased growth rate usingxylose as a sole carbon source for growth, comprising:

-   -   (a) providing yeast cells of the strain as a portion of a        population of genetically diverse yeast cells of Saccharomyces;    -   (b) culturing the population of genetically diverse        non-recombinant yeast cells of Saccharomyces under conditions        which permits combining of DNA between the yeast cells of the        population;    -   (c) screening or selecting yeast cells for derivatives of the        strain that have an increased growth rate on xylose;    -   (d) isolating one or more derivatives of the strain which have        an increased growth rate using xylose as a sole carbon source        for growth relative to the growth rate of the strain using        xylose as a sole carbon source for growth.

The yeast cells are typically screened or selected by incubating theyeast cells on or in xylose-containing medium.

In one embodiment, the yeast cells are selected in step (c). The yeastcells may be selected in step (c) by incubating the yeast cells on or inxylose-containing medium for sufficient time to permit yeast cellscapable of growth on xylose as a sole carbon source to grow using xyloseas the carbon source. The yeast cells may be selected in step (c) byincubating the yeast cells on or in xylose-containing medium forsufficient time to permit yeast cells having an increased growth rateusing xylose as a sole carbon source to grow using xylose as the carbonsource.

In another embodiment, the yeast cells are screened in step (c). Theyeast cells may be screened in step (c) by incubating the yeast cells onor in xylose-containing medium for sufficient time to permit yeast cellshaving an increased growth rate using xylose as a sole carbon source togrow using xylose as the carbon source, and thereafter collecting theyeast cells that grow fastest using the xylose as a carbon source.

Steps (b) and (c) may typically be repeated, whereby the derivativesform at least a portion of the population of genetically diverseSaccharomyces strains, until one or more derivatives have acquired anincreased growth rate on xylose as a sole carbon source for growth.

In the past, to address the problem of the inability of Saccharomycescerevisiae to use xylose, others have used recombinant DNA approaches tointroduce genes obtained from yeast which can utilise xylose as a carbonsource to confer on Saccharomyces the ability to utilise xylose (eg.Sonderegger and Sauer (2003) Applied and Environmental Microbiology 69:1990-1998). In these approaches, the genes for xylose utilisation havebeen cloned from organisms which are capable of utilising xylose forgrowth, and subsequently introduced into Saccharomyces cerevisiae. Forexample, fungal xylose isomerase from Piromyces spp. has been integratedinto the genome of Saccharomyces cerevisiae to generate strains whichgrow slowly on xylose as a sole carbon source (Kuyper et al. 2003).Xylose reductase from Pichia stipitis has also been cloned intoSaccharomyces cerevisiae to generate yeast that can utilise xylose as asole carbon source for growth (Wahlbom et al., 2003).

Non-recombinant Saccharomyces cerevisiae is “generally regarded as safe”(GRAS) and if recombinant techniques are used to develop Saccharomycescerevisiae that utilize xylose, they lose the GRAS status. It istherefore not necessarily industrially or economically useful,desirable, or suitable to use strains of Saccharomyces cerevisiae thatcontain genes from other genera or species, or that are produced usingrecombinant DNA methods. Indeed strains that are derived throughrecombinant DNA approaches are not industrially useful where there aresocio-, or enviro-political or other barriers to use of such strains.

The use of recombinant DNA techniques reduces the opportunities anddesirability to use yeasts in human foods etc., whereas non-recombinantstrains could be advantageously used for human foods etc. The ability touse non-recombinant yeast simultaneously for ethanol and for human foodsprovides opportunities to improve cost-efficiencies of yeast-basedprocesses. For example it is possible to use non-recombinant yeasts toconvert xylose to ethanol and yeast biomass. The ethanol may be used forfuel and other applications whereas the yeast produced may be used inother valuable applications such as production of extracts or otherby-products.

The inventors have found that when non-recombinant wild-type strains ofSaccharomyces are incubated on medium comprising xylose as a sole carbonsource, very slow growth on the xylose is detectable. This is contraryto the prior art, which clearly indicates that Saccharomyces is notcapable of growth on xylose. The inventors believe that the growth ofSaccharomyces on xylose is so slow that it has not been previouslydetected. As described herein, the inventors have found that when solidminimal mineral medium containing xylose as a sole carbon source wasinoculated with wild-type strains of Saccharomyces cerevisiae, growthwas detectable by microscopic examination of colonies followingincubation for 2 months at 30° C. Although the detectable growth allowedapplication of non-recombinant strategies to be used to derive yeastwith improved growth rates, the observed very slow growth rate does nothave industrial utility.

The inventors have extended their finding that Saccharomyces is capableof very slow growth on xylose as a sole carbon source to develop strainsof Saccharomyces that are capable of growth on xylose as a sole carbonsource at much higher rates than strains of Saccharomyces to which themethod of the present invention has not been applied. Prior to theinventors finding, it was not thought possible that incubating yeastcells of Saccharomyces in or on medium containing xylose as sole carbonsource would result in any selection or enrichment of yeast cells whichwere capable of growth on xylose as it was believed that the yeast wouldnot grow in the medium because xylose was seen as a non-useable carbonsource for Saccharomyces. Nevertheless, by using their findings and acombination of selection strategies and methods such as mating ofpopulations of genetically diverse strains of Saccharomyces, suitablystrains of Saccharomyces cerevisiae, the inventors have found thatSaccharomyces strains can be produced that are capable of growth onsolid medium, and/or in liquid medium, containing xylose as a solecarbon source at rates that are higher than those of Saccharomycesstrains to which methods of the present invention have not been applied.Moreover, the inventors have found that by employing selectionstrategies and methods such as mating of genetically diverse strains ofSaccharomyces, Saccharomyces strains can be produced that have a growthrate using xylose as a sole carbon source that is at least Onegeneration per 48 hours, and in some advantageous embodiments, may begreater than one generation per 4 hours. Thus, by using non-recombinantmethods, the inventors have been able to generate Saccharomyces strainsthat are capable of growth in liquid and on solid media containingxylose as a sole carbon source at industrially useful rates.

In a fourth aspect, the invention provides a Saccharomyces strainproduced by the method of the first to third aspects of the invention.

In an fifth aspect, the invention provides an isolated Saccharomycesstrain which is capable of a growth rate of at least one generation per48 hours using xylose as a sole carbon source for growth, wherein thestrain produces:

-   -   (i) a 5-fold increase in biomass when grown under the conditions        specified in Test T1; and    -   (ii) at least 10 mg dry weight of biomass when grown under the        conditions specified in Test T2, and wherein the strain is        produced by the method of the first to third aspects.

In a sixth aspect, the invention provides an isolated Saccharomycesstrain which is capable of a growth rate of at least one generation per48 hours using xylose as a sole carbon source for growth, wherein:

-   -   (i) the strain produces a 10-fold increase in biomass when grown        under the conditions specified in Test T1; and    -   (ii) the strain produces at least 50 mg dry weight of biomass        when grown under the conditions specified in Test T2; and    -   (iii) at least 0.1 g/l of ethanol is detected under the        conditions specified in Test T3; and    -   (iv) at least 1 nanomole of NAD(P)H is reduced or oxidised per        minute per mg of protein extract at 30° C. under the conditions        specified in Test T4; and    -   (v) at least 1 nanomole of NAD(P)H is reduced or oxidised per        minute per mg of protein extract at 30° C. under the conditions        specified in Test T5; and    -   wherein the strain is produced by the method of the first to        third aspects.

In an seventh aspect, the invention provides an isolated Saccharomycesstrain which is capable of a growth rate of at least one generation per48 hours using xylose as a sole carbon source for growth, wherein:

-   -   (i) the strain produces at least a 5-fold increase in biomass        when grown under the conditions specified in Test T1; and    -   (ii) the strain produces at least 40 mg dry weight of biomass        when grown under the conditions specified in Test T2; and    -   (iii) at least 1 nanomole of NAD(P)H is reduced or oxidised per        minute per mg of protein extract at 30° C. under the conditions        specified in Test T4; and    -   (iv) at least 1 nanomole of NAD(P)H is reduced or oxidised per        minute per mg of protein extract at 30° C. under the conditions        specified in Test T5; and    -   (v) the strain produces at least a 5-fold increase in biomass        under the conditions specified in Test T7;    -   (vi) a concentration of at least 0.04 g/l of ethanol is detected        under the conditions specified in Test T8;    -   and wherein the strain is produced by the method of the first to        third aspects.

In one embodiment of the fourth to seventh aspects, the strain producesat least 0.2 grams of ethanol per liter within a period of 4 monthsunder the conditions specified in Test T9.

In an eighth aspect, the invention provides an isolated Saccharomycesstrain that is capable of growing at a rate of at least one generationper 48 hours using xylose as a sole carbon source for growth, whereinthe capability of the strain to utilise xylose as a sole carbon sourceis obtained by non-recombinant methods.

The rate of growth of the strain may be any rate that is at least onegeneration per 48 hours. The rate of growth may be at least onegeneration per 36 hours. The rate of growth may be greater than onegeneration per 24 hours.

The rate of growth may be greater than one generation per 12 hours. Therate of growth may be greater than one generation per 10 hours. The rateof growth may be greater than one generation per 8 hours. The rate ofgrowth may be greater than one generation per 6 hours.

The rate of growth may be greater than one generation per 4 hours. Therate of growth may be greater than one generation per 2 hours. The rateof growth may be greater than one generation per 80 minutes.

In one embodiment, the strain is capable of growth using xylose as asole carbon source at a rate that is substantially the same as the rateof growth of the strain using glucose as a sole carbon source forgrowth.

In one embodiment, the strain has a growth rate of at least onegeneration per 48 hours on xylose minimal mineral medium.

In one embodiment, the strain produces a 2-fold increase in biomass whengrown under the conditions specified in Test T1. Typically, the strainproduces at least a 5 fold increase in biomass when grown under theconditions specified in Test T1. Suitably, the strain produces at leasta 10-fold increase in biomass when grown under the conditions specifiedin Test T1.

In one embodiment, the strain produces at least 0.01 g dry weight ofbiomass per 50 ml of culture when grown under the conditions specifiedin Test T2.

In various embodiments: (i) the strain expresses a non-recombinantenzyme having at least 1 unit of xylose reductase activity under theconditions specified in Test T4;

(ii) the strain expresses a non-recombinant enzyme having at least 1unit of xylitol dehydrogenase activity under the conditions specified intest T5;

(iii) the strain expresses a non-recombinant enzyme having at least oneunit of xylose reductase activity under the conditions specified in testT4, a non-recombinant enzyme having at least one unit of xylitoldehydrogenase activity under the conditions specified in test T5.

In a ninth aspect, the invention provides an isolated Saccharomycesstrain which is capable of a growth rate of at least one generation per48 hours using xylose as a sole carbon source for growth, and which iscapable of expressing non-recombinant enzyme having an activity selectedfrom the group consisting of xylose reductase and xylitol dehydrogenase,wherein the xylose reductase activity is at least 1 unit when determinedby test T4, and the xylitol dehydrogenase activity is at least 1 unitwhen determined under the conditions specified in test T5.

The strain may be capable of expressing a non-recombinant enzyme havingxylose reductase activity and a non-recombinant enzyme having xylitoldehydrogenase activity. In one embodiment, the strain is capable ofexpressing a non-recombinant enzyme having xylulose kinase activity inaddition to one or more non-recombinant enzymes having an activityselected from the group consisting of xylose reductase and xylitoldehydrogenase. Typically, the xylulose kinase activity is at least 5units when determined by test T6.

Typically, the strain of the ninth aspect has a rate of growth of atleast one generation per 48 hours using xylose as a sole carbon source.

In one embodiment, the strain produces at least a 2-fold increase inbiomass when grown under the conditions specified in Test T1. Typically,the strain produces at least a 5-fold increase in biomass when grownunder the conditions specified in Test T1. More typically, the strainproduces at least a 5-fold increase in biomass when grown under theconditions specified in test T1.

The strain may produce at least 10 mg dry weight of biomass under theconditions specified in test T2. The strain may produce at least 30 mgdry weight of biomass under the conditions specified in test T2. Thestrain may produce at least 40 mg dry weight of biomass under theconditions specified in Test T2. Typically, the strain produces at least50 mg dry weight of biomass under the conditions specified in test T2.

The strain may produce at least 0.01 g dry weight of biomass per 50 mlof culture when grown under the conditions specified in Test T2.

The Saccharomyces strain may further be capable of using xylitol as asole carbon source for growth. Typically, the capability of the strainto utilise xylitol as a sole carbon source is obtained throughnon-recombinant methods.

In one embodiment, the strain produces at least a 5-fold increase inbiomass when grown under the conditions specified in Test T7.

The Saccharomyces strain may be capable of aerobic or anaerobic growthusing xylose as a sole carbon source. Typically, the growth is aerobicgrowth. However, the Saccharomyces strain may also grow anaerobically ormicroaerophilically using xylose as a sole carbon source.

The Saccharomyces strain may be capable of growth on glucose underanaerobic conditions.

The Saccharomyces strain may further be capable of utilising xylose toproduce one or more of a carbon-based compound. Examples of suitablecarbon-based compounds include alcohols, xylitol, organic acids such asacetic acid, glycerol, carbon dioxide, or other yeast components,metabolites and by-products including yeast extracts, proteins,peptides, amino acids, RNA, nucleotides, glucans, etc. Typically, thealcohol is ethanol.

The strain may produce ethanol when growing on xylose. The strain mayuse the xylose to produce ethanol without growing on the xylose. Thestrain may produce ethanol from xylose. Typically, the strain fermentsxylose to produce ethanol.

The strain may produce ethanol at a concentration of at least 0.1 g/Lunder the conditions specified in Test T3.

The strain may produce ethanol at a concentration of at least 0.4 g/Lunder the conditions specified in Test T8.

The strain may produce at least 0.2 g of ethanol per liter within aperiod of 4 months under the conditions specified in Test T9.

The strain may produce at least 0.5 g of ethanol per L when inoculatedat a cell density of at least 5×10⁸ cells per ml into xylose-containingminimal mineral medium. The xylose-containing minimal mineral medium maycontain glucose. Typically, the strain produces 0.5 g/L of ethanolwithin 5 hours of inoculation of the medium.

In one embodiment, the strain ferments xylose to produce at least 0.05grams of ethanol per liter of culture under the conditions specified inTest T3.

The strain may be capable of utilising xylose as a sole carbon source togrow on solid medium, and/or in liquid medium. In one embodiment, thestrain is capable of growth in liquid medium containing xylose as a solecarbon source. The liquid medium may be liquid mineral medium containingxylose as a sole carbon source.

The Saccharomyces strain may be a strain from any species of the genusSaccharomyces. Examples of suitable species of yeasts include S.cerevisiae, S. paradoxus, S. mikatae, S. cariocanus, S. kudriavzevii, S.pastorianus and S. bayanus. Typically the species is Saccharomycescerevisiae. Typically the strain will be capable of mating withSaccharomyces of the same species. Typically the strain will be capableof mating with Saccharomyces cerevisiae. Typically, the strain isSaccharomyces cerevisiae.

In one embodiment of the eighth or ninth aspects, the Saccharomycesstrain is recombinant.

In a tenth aspect, the invention provides an isolated non-recombinantSaccharomyces strain which produces an increase in biomass of at least2-fold under the conditions specified in Test T1.

In one embodiment, the increase in biomass is at least 5-fold, typicallyat least 10-fold.

In a eleventh aspect, the invention provides an isolated non-recombinantSaccharomyces strain which has the following characteristics:

-   -   (a) Biomass of the strain increases at least 5-fold under the        conditions specified in test T1;    -   (b) At least 10 mg dry weight of biomass is produced under the        conditions specified in test T2.

In a twelfth aspect, the invention provides an isolated non-recombinantSaccharomyces strain which has the following characteristics:

-   -   (a) Biomass of the strain increases at least 10-fold under the        conditions specified in test T1;    -   (b) At least 50 mg dry weight of biomass is produced under the        conditions specified in test T2;    -   (c) A concentration of at least 0.1 g/L of ethanol is detected        under the conditions specified in Test T3;    -   (d) at least 1 nanomole of NAD(P)H is reduced or oxidised per        minute per mg of protein extract at 30° C. under the conditions        specified in Test T4; and    -   (e) at least 1 nanomole of NAD(P)H is reduced or oxidised per        minute per mg of protein extract at 30° C. under the conditions        specified in Test T5.

In a thirteenth aspect, the invention provides an isolatednon-recombinant Saccharomyces strain which has the followingcharacteristics:

-   -   (a) Biomass of the strain increases at least 5-fold under the        conditions specified in test T1;    -   (b) At least 40 mg dry weight of biomass is produced under the        conditions specified in test T2;    -   (c) Biomass of the strain increases at least 5-fold under the        conditions specified in Test T7;    -   (d) A concentration of at least 0.04 g/L of ethanol is detected        under the conditions specified in test T8;    -   (e) at least 1 nanomole of NAD(P)H is reduced or oxidised per        minute per mg of protein extract at 30° C. under the conditions        specified in Test T4; and    -   (f) at least 1 nanomole of NAD(P)H is reduced or oxidised per        minute per mg of protein extract at 30° C. under the conditions        specified in Test T5.

Examples of suitable Saccharomyces strains that are capable of growth ata rate of at least one generation per 48 hours using xylose as a solecarbon source for growth are those that are deposited under the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure at the Australian GovernmentAnalytical Laboratories, 1 Suakin Street, Pymble, NSW 2073, Australia(now known as the National Measurement Institute (NMI), which is also anInternational Depository Institution under the Budapest Treaty, havingthe address: 1/153 Bertie Street, Port Melbourne, VIC 3207), underdeposit accession nos. NM04/41257, NM04/41258, NM05/45177 (ISO 10) andNM05/45178 (ISO 7). Deposit accession numbers NM04/41257 and NM04/41258were deposited on 12 May 2004. Deposit accession numbers NM04/45177 andNM05/45178 were deposited on 16 May 2005. All restrictions imposed bythe depositor on the availability to the public of the depositedmaterial will be irrevocably removed upon the granting of a patent.

The Saccharomyces strain may be obtained by any method referred toherein in which the ability to utilise xylose as a sole carbon sourcefor growth is obtained by non-recombinant methods. Methods that may beemployed to obtain the strain include combinations of, naturalselection, mating procedures, mutagenesis, or other so-called classicalgenetic methods known to those skilled in the art and discussed andreferred to in, for example, Attfield and Bell (2003)“Genetics andclassical genetic manipulations of industrial yeasts” in, Topics inCurrent Genetics, Vol 2. Functional Genetics of Industrial Yeasts (J. H.de Winde, ed.), Springer-Verlag Berlin Heidelberg or combinationsthereof.

The ability to grow at a rate of at least one generation per 48 hoursusing xylose as a sole carbon source is typically obtained by matingbetween Saccharomyces strains and screening or selection for increasedgrowth rate using xylose as a sole carbon source.

For example, the strain may be obtained by conducting rare or directedor mass matings between strains of sexually compatible Saccharomyces,followed by selection for strains that are capable of utilising xyloseas a sole carbon source. The strain may be derived from one or morenaturally occurring isolates of Saccharomyces, spontaneously mutatedisolates, or it May be obtained by exposing one or more Saccharomycesstrains to a mutagen and subsequently used in mating and selectionstrategies to screen or select for a mutant strain that is capable ofutilising xylose as a sole carbon source. The obtained strain may beselected by any of the selection methods referred to herein.

In a fourteenth aspect, the invention provides a derivative of a strainof the fourth to thirteenth aspects.

Methods for the production of derivatives of yeast strains are wellknown in the art and include matings with other yeast strains, cellfusions, mutagenesis, and/or recombinant methods.

In a fifteenth aspect, the invention provides the use of a Saccharomycesstrain of the fourth to fifteenth aspects, or a derivative of thefourteenth aspect, in production of yeast biomass. The yeast biomass maybe used in, for example, the baking industry, for biomass products.

In a sixteenth aspect, the invention provides the use of a Saccharomycesstrain of the fourth to thirteenth aspect, or a derivative of thefourteenth aspect, for the production of ethanol from xylose.

In a seventeenth aspect, the invention provides a method for theproduction of ethanol comprising incubating a Saccharomyces strain ofthe fourth to thirteenth aspects, or a derivative of the fourteenthaspect, with a xylose-containing medium, under conditions which permitthe strain to ferment xylose to produce ethanol.

In an eighteenth aspect, the invention provides a method of convertingxylose into yeast biomass, comprising culturing a Saccharomyces strainof the fourth to thirteenth aspects, or a derivative of the fourteenthaspect, with a xylose-containing medium under conditions which permitthe strain to grow using the xylose as a carbon source for growth.

In a nineteenth aspect, the invention provides a method of producingyeast biomass comprising growing a Saccharomyces strain on or in axylose-containing medium wherein at least a portion of the yeast biomassis produced using xylose as a carbon source for growth.

In a twentieth aspect, the invention provides a method of producing acompound from a Saccharomyces strain, comprising culturing theSaccharomyces strain on or in a xylose-containing medium underconditions which permit production of the compound, wherein the compoundis produced by the strain using xylose as a carbon source.

In one embodiment, the compound is produced by the strain during growthusing xylose as a carbon source.

In one embodiment, the method comprises the further step of recoveringthe compound.

The compound may be any compound that can be produced by theSaccharomyces strain. Examples of suitable compounds include ethanol,CO₂, enzymes, recombinant enzymes, recombinant proteins, yeastby-products, vitamins, nucleotides, ribonucleotides,deoxyribonucleotides, lipids, yeast proteins, xylitol.

It will be appreciated by those skilled in the art that growth of theyeast cells produces biomass, and therefore any method which results ingrowth will result in production of biomass. Thus, for example,production of ethanol from growth on xylose-containing medium will inaddition produce biomass. In one embodiment, compounds are containedwithin the yeast cells in biomass and the compounds may be recoveredfrom the yeast cells using methods well known in the art for extractingcompounds from yeast cells.

In a twenty-first aspect, the invention provides a compound produced bythe method of the eighteenth aspect.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a plot of the average doubling time of a population ofSaccharomyces cerevisiae strains over time following application of themethod of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention employs, unless otherwiseindicated, conventional microbiology and classical genetics. Suchtechniques are known to the skilled worker, and are explained fully inthe literature. See, for example, Sherman et al. “Methods in YeastGenetics” (1981) Cold Spring Harbor Laboratory Manual, Cold SpringHarbor, N.Y.; European Patent number EP 0 511 108 B.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. It must be noted that as used herein and in theappended claims, the singular forms “a”, “an”, and “the” include pluralreference unless the context clearly indicates otherwise. Thus, forexample, a reference to “a cell” includes a plurality of such cells.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any materials andmethods similar or equivalent to those described herein can be used topractice or test the present invention, preferred materials and methodsare now described.

All publications mentioned herein are cited for the purpose ofdescribing and disclosing the protocols and reagents which are reportedin the publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Xylose is a sugar which is inexpensive, can be obtained from a renewableresource, and is available in large amounts. Xylose can represent asignificant portion of hemicellulosic plant biomass. Plant biomassincludes agricultural residues, paper wastes, wood chips and the like,and is renewable, and available at low cost in large amounts. Xylose isprimarily present in hemicellulosic plant biomass as polymers known asxylan and hemicellulose. The polymers of xylose can be readily brokendown into monomeric sugars either by chemical means such as acidhydrolysis, or by enzymatic means using enzymes such as xylanases. Inpaper manufacturing for example, xylose is one of the main sugars in thewaste stream, where it contributes to biological oxygen demand (BOD) andthereby makes disposal of the waste-water environmentally difficult. Forevery kilogram of paper produced from hardwood, typically 100 grams ofsugar is produced, 35 grams of which is xylose. Because of itsabundance, xylose presents a major potential carbon source for producingyeast biomass and by-products of yeast metabolism such an ethanol.

In one aspect there is provided to a Saccharomyces strain that iscapable of growing at a rate of at least one generation per 48 hoursusing xylose as a sole carbon source. The inventors have found that itis possible to obtain strains of Saccharomyces that are capable ofrelatively rapid growth using xylose as a sole carbon source without theneed to use recombinant DNA technology. This is an unexpected result asit was previously believed that strains of Saccharomyces were notcapable of growth on xylose as a sole carbon source. Until the presentinvention, Saccharomyces strains capable of growth at rates of at leastone generation per 48 hours using xylose as a sole carbon source haveonly be obtained by introducing into Saccharomyces strains cloned xyloseutilisation genes from other organisms that are capable of growth onxylose. Alternatively unnaturally occurring combinations of clonedSaccharomyces gene promoters and cloned Saccharomyces DNA sequences havebeen generated using recombinant methods and introduced intoSaccharomyces strains. Thus, prior to the present invention, obtainingstrains of Saccharomyces capable of utilising xylose as a sole carbonsource has not been possible unless either xylose utilisation genes fromother organisms that are capable of growth on xylose have been clonedand introduced into the Saccharomyces strain, or strong Saccharomycesgene promoters have been operably linked to other Saccharomyces DNAsequences and introduced into the Saccharomyces strain using recombinantDNA methods.

Prior to the present invention, no non-recombinant yeast from the genusSaccharomyces were capable of a growth rate of at least one generationper 48 hours using xylose as a sole carbon source. U.S. Pat. No.4,511,656 discloses yeast strain ATCC No. 20618 which is anon-recombinant yeast mutant that is capable of producing ethanol whenincubated in medium containing xylose. However, ATCC 20618 is not fromthe genus Saccharomyces. The morphology of the colonies produced by ATCCNo. 20618 is not consistent with those produced by Saccharomyces. ATCCNo. 20618 does not sporulate, does not mate with standard strains ofSaccharomyces cerevisiae, and sequencing of the ITS regions and histoneH3-H4 intergenic region of ATCC No 20618 reveals that it is not of thegenus Saccharomyces, but is closely related to Candida tropicalis.Accordingly, ATCC 20618 is phylogenetically distant from Saccharomycesas defined by Kurtzman (2003) FEMS Yeast Research 4:233-245 and asdefined herein. Candida tropicalis is well known to utilise xylose.Additionally, the mutants disclosed in U.S. Pat. No. 4,511,656 were onlydemonstrated to produce ethanol when provided with growth nutrients suchas yeast extract, malt extract and peptone in the growth medium. Thesegrowth nutrients provide alternative fermentable carbon sources toxylose. Thus, U.S. Pat. No. 4,511,656 does not disclose strains ofSaccharomyces that are capable of growth on, or fermentation of, xyloseas a sole carbon source.

Furthermore U.S. Pat. No. 4,511,656 teaches that yeast capable ofproducing ethanol are selected which form colonies on xylose but notxylitol. Without wishing to be bound by theory, the inventors believethat contrary to the teaching of U.S. Pat. No. 4,511,656, the metabolismof xylitol, which is an intermediate in xylose utilisation, might beexpected to be advantageous to growth on xylose and ethanol productionfrom xylose.

The inventors have found contrary to the teaching of the prior art, thatnot only are Saccharomyces strains capable of very slow growth on xyloseas a sole carbon source, but that the growth rate using xylose as a solecarbon source of Saccharomyces strains can be increased withoutintroducing cloned xylose utilisation genes.

In one aspect, there is provided an isolated Saccharomyces strain whichis capable of growing at a rate of at least one generation per 48 hoursusing xylose as a sole carbon source. As used herein, a Saccharomycesstrain is a strain of the genus Saccharomyces as defined in Kurtzman(2003) FEMS Yeast Research vol. 4 pp. 233-245.

As used herein, the expression “capable of growing at a rate of at leastone generation per 48 hours using xylose as a sole carbon source forgrowth” means that the strain is capable of using xylose as the onlysource of carbon for generation of energy, and synthesis of moleculesthat are necessary for growth of the strain, to attain a growth rate ofat least one generation per 48 hours. The term “one generation” will beclear to those skilled in the art to mean one cycle of cell division.The strain will preferably be capable of growth in solid and liquidmedia in which xylose is the only carbon source. It will be appreciatedby persons skilled in the art that a strain that is capable of growingat a rate of at least one generation per 48 hours using xylose as a solecarbon source will be capable of growth on solid and/or in liquidminimal mineral media, in which xylose is the sole carbon source. Anexample of a suitable liquid medium is the commercially available DIFCOLaboratories Yeast Nitrogen Base without amino acids, which contains allessential inorganic salts and vitamins necessary for cultivation ofyeasts except a source of carbohydrate or amino acids, supplemented withbetween 0.01% and 50% xylose, typically 1% and 10% xylose, suitably 5%xylose. Typically, solid medium is liquid medium that has beensolidified by the addition of a gelling agent such as agar. It will alsobe appreciated by persons skilled in the art that a strain that iscapable of growth using xylose as a sole carbon source may also becapable of growth on many other sugars. The capability of the strain toutilise xylose as a sole carbon source is obtained by non-recombinantmethods. As used herein, the expression “non-recombinant methods” referto any methods that do not utilise recombinant DNA technology to producein the Saccharomyces strain the ability to utilise xylose. In otherwords, the genes that confer on the strain the ability to utilise xylosefor growth have not been introduced into the strain using recombinantmethods. As used herein, a “recombinant method” is a method in which oneor more genes are introduced into an organism using recombinant DNAtechniques. As used herein, recombinant DNA techniques refer totechniques in which genetic information is manipulated in vitro, such aswhen genes are isolated and cloned from an organism. Thus,non-recombinant methods are methods which do not involve themanipulation of genetic information in vitro. A non-recombinant strainis a strain into which recombinant nucleic acid has not been introduced.

Non-recombinant methods may include, for example, mutagenesis, classicalmating, cytoduction, cell fusion such as protoplast fusion, andcombinations thereof. The non-recombinant method may comprise culturinga population of genetically diverse yeast cells of Saccharomyces underconditions which permit combining of the DNA between yeast cells invivo, and screening or selecting for yeast cells having an increasedgrowth rate using xylose as a sole carbon source, typically byincubating a genetically diverse population of yeast cells in or onxylose-containing medium for sufficient time to select yeast cellshaving an increased growth rate using xylose as a sole carbon source.

It will be understood by those skilled in the art that the geneticinformation which confers the ability of the Saccharomyces strain togrow at a rate of at least one generation per 48 hours utilising xyloseas a sole carbon source is obtained from the genus Saccharomyces. Inother words, all the genetic information necessary for growth at a rateof at least one generation per 48 hours is obtained from within the genepool of the genus Saccharomyces. Typically, the genetic informationwhich confers the ability of the Saccharomyces strain to grow at a rateof at least one generation per 48 hours utilising xylose as a solecarbon source for growth is obtained from a genetically diversepopulation of non-recombinant Saccharomyces strains. As discussed above,prior to the present invention, the gene pool of the genus Saccharomyceswas thought not to contain the genetic information for conferring theability to utilise xylose as a sole carbon source for growth.

In another aspect, there is provided isolated Saccharomyces strainswhich have the ability to grow on xylose as a sole carbon source at agrowth rate of at least one generation per 48 hours, and which arecapable of expressing non-recombinant enzyme having an activity selectedfrom the group consisting of xylose reductase and xylitol dehydrogenase.The enzyme has an activity of at least unit as determined by Test T4 forxylose reductase, and Test T5 for xylitol dehydrogenase. Tests T4 and T5are defined herein. In one embodiment, the xylose reductase activity isat least 1.5 units, suitably at least 3 units. In one embodiment, thexylitol dehydrogenase activity is at least 5 units, suitably at least 8units.

There is also provided a method for producing a Saccharomyces strainthat is capable of growth at a desired growth rate using xylose as asole carbon source. The “desired growth rate” may be any growth ratewhich is greater than the growth rate of the yeast cells ofSaccharomyces prior to applying the method of the invention. The desiredgrowth rate will be greater than the growth rate of Saccharomycescerevisiae strain NL67 on xylose as a sole carbon source. The desiredgrowth rate may be at least one generation per 48 hours, suitablygreater than one generation per 24 hours, suitably greater than onegeneration per 12 hours, typically greater than one generation per 10hours, more typically greater than one generation per 8 hours, and maybe as high as the rate of growth of Saccharomyces on glucose, which isabout one generation per 80 minutes.

The method comprises providing a population of genetically diversenon-recombinant yeast cells of Saccharomyces. As used herein, theexpression “genetically diverse non-recombinant yeast cells” refers toat least two non-recombinant yeast cells that have distinct genotypes.The genetically diverse non-recombinant yeast cells may be a mixture ofyeast cells of different Saccharomyces strains obtained from the wild,from wine, distilling and beer fermentation, from baking applications,or from any other source of Saccharomyces. The genetically diversepopulation of non-recombinant yeast cells of Saccharomyces may bederived from a single strain that is sporulated to derive geneticallydiverge progeny. The genetically diverse population of non-recombinantyeast cells of Saccharomyces may include one or more yeast cells ofSaccharomyces strains that have been exposed to a physical or chemicalmutagen such as, for example, ultraviolet light, X-ray or Gamma ray, orethylmethanesulphonate, nitrosoguanidine, mitomycin C, bleomycin, or anyother agents that cause alterations to DNA base sequences. Thus,genetically diverse non-recombinant yeast cells includes within itsscope yeast cells of strains that have been generated by mutagenesis.Methods for mutagenesis of yeast, and in particular, mutagenesis ofSaccharomyces cerevisiae, are known to persons skilled in the art andare described in, for example, Sherman et al. “Methods in YeastGenetics” (1981) Cold Spring Harbor Laboratory Manual, Cold SpringHarbor, N.Y. The genetically diverse population of non-recombinant yeastcells of Saccharomyces may also include yeast cells with spontaneouslyoccurring mutations. The population of genetically diversenon-recombinant yeast cells of Saccharomyces may be all the samespecies, or may be different species of Saccharomyces. Typically, thedifferent species are capable of mating between each other. For example,the population of genetically diverse non-recombinant yeast cells maycomprise different strains of Saccharomyces cerevisiae, or any otherSaccharomyces species, obtained from any of the sources mentioned above.Examples of suitable Saccharomyces species include S. cerevisiae, S.paradoxus, S. mikatae, S. cariocanus, S. kudriavzevii, S. pastorianusand S. bayanus. Typically, the population of genetically diverse yeastcells of Saccharomyces are the same species. Typically, the species isSaccharomyces cerevisiae.

Examples of yeasts that could be used to provide starting inocula forpopulations of genetically diverse non-recombinant yeast cells includeSaccharomyces straits available from well known culture collections suchas American Type Culture Collection (ATCC), e.g. ATCC 4111, ATCC 26603,ATCC 38559, the National Collection of Yeast Cultures (NCYC) e.g. S.cerevisiae NCYC 995, NCYC 996, the Central Bureau voor Schimmel Cultures(CRBS) e.g. S. cerevisiae CBS 745.95, CBS 755.95, or isolated fromcommercially available yeasts sold by any number of companies for use intraditional formulations such as baking, brewing, wine, distilling, etc.

The population of genetically diverse non-recombinant yeast cells ofSaccharomyces are cultured under conditions which permit combining ofDNA between the yeast cells. Combining of DNA between the yeast cellsmay be by any method suitable for combining DNA from at least two yeastcells, provided the method is not a recombinant method. Examples ofsuitable methods for combining the DNA of yeast cells include mating andcytoduction. As used herein, the term “mating” refers to the process ofexchange and recombination of DNA between at least two yeast cells,including by classical genetic mating methods, directed mating, massmating, rare mating, cell fusion, etc. For example, classical geneticcrosses or cell fusion may be used to generate intraspecific orinterspecific hybrids.

In one embodiment, the yeast cells are cultured under conditions whichpermit mating between the yeast cells. Typically, the yeast cells arecultured under conditions which permit mating by sporulating the yeastcells and thereafter mating the sporulated yeast. For example, the yeastcells may be mated by:

-   -   (a) sporulating the yeast cells;    -   (b) germinating the sporulated yeast cells;    -   (c) hybridising compatible mating types of the sporulated yeast        cells.

Typically, the yeast cells are mated by:

-   -   (a) pooling the population of genetically diverse        non-recombinant yeast cells;    -   (b) sporulating the pooled cells and germinating the spores to        produce haploid cells;    -   (c) hybridisation of compatible mating types of the haploid        cells to produce hybrid yeast cells.

Methods for sporulation, obtaining haploids and hybridisation of thehaploids to produce hybrid yeast cells are known in the art and aredescribed in, for example, Chapter 7, “Sporulation and Hybridisation ofYeast” by R. R. Powell, in “The Yeasts” vol 1 edited by A. H. Rose andJ. S. Harrison, 1969, Academic Press; EP 0 511 108 B. Typically, themating is mass mating. Mass mating involves culturing at least two andtypically millions of different yeast cells together in a manner whichpermits mating to occur between compatible mating types. Methods formass mating are described in, for example, Higgins et al. (2001),Applied and Environmental Microbiology vol. 67, pp. 4346-4348; Lindegren(1943), Journal of Bacteriology vol. 46 pp. 405-419.

In another embodiment, the yeast are cultured under conditions whichpermit cell fusion. Methods for the generation of intraspecific orinterspecific hybrids using cell fusion techniques are described in, forexample, Morgan (1983) Experientia suppl. 46: 155-166; Spencer et al.(1990) in, Yeast Technology, Spencer J F T and Spencer D M (eds.),Springer Verlag New York. Cytoduction methods are described in, forexample, Inge-Vechtomov et al. (1986) Genetika 22: 2625-2636; Johnston(1990) in, Yeast Technology, Spencer J F T and Spencer D M (Eds.),Springer Verlag New York; Polaina et al. (1993) Current Genetics 24:369-372.

The yeast cells are screened or selected for yeast cells which have anincreased growth rate using xylose as a sole carbon source for growth.The yeast cells are screened or selected to enrich or identify yeastcells which have an increased rate of growth using xylose as a solecarbon source. The yeast cells are screened or selected typically byincubating the yeast cells on or in a xylose-containing medium. Theyeast cells are screened or selected for those yeast cells that have animproved ability to grow using xylose as a sole carbon source forgrowth. The improved ability will typically be an increase in growthrate using xylose as a sole carbon source towards the desired growthrate. An increase in growth rate is an increase in the growth rate of ayeast cell relative to the growth rate of cells prior to culturing theyeast cells under conditions which permit combining of DNA between yeastcells. In one embodiment, the yeast cells are selected. The terms“selected” or “selecting” refers to a process in which the yeast cellsthat have an improved ability to grow using xylose as a sole carbonsource for growth at the desired growth rate become enriched in thepopulation. The yeast cells may be selected by incubating the yeastcells on or in xylose-containing medium for sufficient time to permityeast cells capable of growth on xylose as a sole carbon source to growusing xylose as the carbon source, and thereafter collecting the yeastcells that grow. The inventors have found that by incubating the yeastcells for sufficient time to allow even slow growing yeast to grow usingxylose as a carbon source, and thereafter collecting the yeast cellsthat grow, including the slow growing yeast cells, the faster growingyeast cells will represent a greater proportion of the collected yeastcells and therefore be enriched in the collected yeast cells, butgenetic diversity of the yeast cell population is substantiallymaintained by also including slow growing yeast cells. For example, byplating mated yeast cells on solid minimal media containing xylose as asole carbon source, cells capable of more rapidly utilising xylose as asole carbon source would generate larger colonies, and as a consequence,those cells would be selected for and the desired genotype(s) enrichedin the population. However, by also collecting the smaller colonies, thegenetic diversity of the population would be substantially maintainedfor the next repeat of steps (b) and (c). Thus, by incubating the yeastcells on xylose-containing medium for sufficient time to permit theyeast cells to grow using xylose as a carbon source, it is possible tonot only select for those yeast cells with increased growth rate onxylose, but to also maintain the genetic diversity of the population forrepeated cycles of steps (b) and (c). Enhanced selection may be appliedby reducing the time the yeast cells are incubated on thexylose-containing medium such that only yeast cells which grow using thexylose as a carbon source within a preselected time period are selected.Yeast cells of this type may be selected by incubating the yeast cells,on or in the xylose-containing medium for sufficient time to permityeast cells having an increased growth rate using xylose as a solecarbon source to grow using xylose as the carbon source, and thereaftercollecting the yeast cells that grow. Using this approach, greaterselective pressure is placed on the yeast cells to grow at an increasedrate, but as there is likely to be insufficient time for a portion ofthe slow growing yeast cells to grow, some genetic diversity may be lostfor further repeats of steps (b) and (c).

In another embodiment, the yeast cells are screened. As used herein, theterms “screened” or “screening” refers to a process in which the yeastcells that have an improved ability to grow using xylose as a solecarbon source for growth at the desired growth rate are firstidentified, and subsequently isolated. The yeast cells may be screenedby incubating the yeast cells on or in xylose-containing medium forsufficient time to permit yeast cells having an increased growth rateusing xylose as a sole carbon source to grow using xylose as the carbonsource, and thereafter collecting the yeast cells that grow fastestusing the xylose as a carbon source. For example, yeast cells that havean improved ability to grow using xylose as a sole carbon source may beidentified on rich xylose-containing medium as those cells that growfaster and therefore form larger colonies than the yeast cells do nothave an improved ability to grow using xylose as a sole carbon source.The larger colonies that appear on the plate can therefore be isolatedin preference to the smaller colonies to thereby isolate yeast cellsthat have an improved ability to grow using xylose as a sole carbonsource for growth at a desired growth rate.

As discussed above, the yeast cells are screened or selected typicallyby incubating the yeast cells on or in a xylose-containing medium. A“xylose-containing medium” may be any medium which contains xylose andprovides a selective advantage to those yeast cells which have anability to grow using xylose as a sole carbon source more efficiently orfaster than other strains. As used herein; a “selective advantage” isthe ability of a cell or strain to undergo a greater number of celldivisions than other cells or strains because of some attribute, in thiscase, the ability to grow efficiently using xylose. Thus, a medium thatprovides a selective advantage to a strain will permit that strain togrow faster in that medium compared to other strains to which the mediumdoes not provide a selective advantage. It will be appreciated by thoseskilled in the art that yeast can be grown on a wide range of media thatinclude at least one source of carbon, a source of nitrogen, a source ofphosphorus, a source of sulphate, trace elements and vitamins. Thexylose-containing medium may be a minimal media or a complex media. Inone embodiment, the xylose containing medium is a minimal medium. Asuitable minimal medium may comprise, for example, DIFCO Yeast NitrogenBase Formulae (see Difco manual “Dehydrated culture media and reagentsfor microbiology” 10^(th) Edition, Difco Labs 1984), together withxylose at a concentration of between 0.1% and 50%, typically 2% and 15%,more typically 5%. In another embodiment, the xylose-containing mediummay be a complex medium. The concentration of xylose in the complexmedium may be between 0.1% and 50%, typically between 2% and 30%, moretypically between 2% and 15%. In one embodiment, the complex medium iscomplete rich media. An example of complete rich medium is medium whichcomprises yeast extract at between 0.5 and 2%, preferably 0.5%, peptoneat between 0.5% and 2%, preferably 1%, and xylose at a concentration ofbetween 0.5% and 50%, typically 2% and 15%, more typically 5%. Inanother embodiment, the complex medium is selected from the groupconsisting of molasses, starch hydrolysates, cellulosic orhemicellulosic biomass hydrolysate (such as bagasse, stover, wood pulp,straw, waste paper, etc), and combinations thereof, optionallysupplemented with xylose, and/or nutrients. It will be appreciated bypersons skilled in the art that solid medium may be any of theabovementioned media typically supplemented with between 1% and 10%agar, more typically between 1% and 5% agar, still more typicallybetween 1% and 2% agar.

The yeast cells may be screened or selected by incubating the cells onsolid and/or liquid medium. In one embodiment, the yeast cells arescreened or selected by incubating the cells on solid medium. In anotherembodiment, the yeast cells are screened or selected by incubating theyeast cells in liquid medium. In a preferred embodiment, the yeast cellsare screened or selected by incubating the yeast cells first on solidmedium, and subsequently on liquid medium. For example, the method maycomprise:

-   -   (a) providing a population of genetically diverse        non-recombinant yeast cells of Saccharomyces;    -   (b) culturing the yeast cells under conditions to permit mating        of the yeast cells;    -   (c) screening or selecting the yeast cells by incubating the        yeast cells on a solid xylose-containing medium;    -   (d) repeating steps (b) and (c) with the screened or selected        cells forming the population of genetically diverse        non-recombinant yeast cells of Saccharomyces, until one or more        yeast cells have acquired the ability to grow at the desired        growth rate using xylose as a sole carbon source for growth.

Typically, steps (b) and (c) are repeated until one or more yeast cellshave acquired a growth rate on xylose that is sufficient to permit thecells to be conveniently grown in liquid medium containing xylose as asole carbon source. For example, the growth rate on solid minimalmineral medium containing 2% to 5% w/v xylose at which a strain may betransferred to growth in liquid minimal mineral medium containing 2% to5% w/v xylose as a sole carbon source will typically be reachedfollowing at least 2 repeats of steps (b) and (c). More typically atleast 5 repeats of steps (b) and (c). Even more typically, at least 10repeats of steps (b) and (c). It will be appreciated by those of skillin the art that the time at which the yeast cells may be selected orscreened by incubating in liquid medium will vary depending on thepopulation and can readily be determined by incubating a small portionof the population in liquid medium at each repeat of steps (b) and (c).Thereafter, the method may comprise:

-   -   (a) providing a population of genetically diverse        non-recombinant yeast cells of Saccharomyces;    -   (b) culturing the yeast cells under conditions to permit mating        of the yeast cells;    -   (c) screening or selecting the yeast cells by incubating the        yeast cells in liquid xylose-containing medium;    -   (d) repeating steps (b) and (c) with the screened or selected        cells forming the population of genetically diverse        non-recombinant yeast cells of Saccharomyces, until one or more        yeast cells have acquired the ability to grow at the desired        growth rate using xylose as a sole carbon source for growth.

The yeast cells are typically incubated on or in the xylose-containingmedium for sufficient time to allow the yeast cells capable of growthusing xylose as a sole carbon source to grow. As discussed above, thelength of time will at least be a sufficient length of time to allow theyeast cells that have an increased rate of growth using xylose as a solecarbon source to grow. Typically, the length of time will be asufficient length of time to permit growth of yeast cells that do nothave an increased rate of growth using xylose as a sole carbon source.In other words, the length of time will be sufficient to permitsubstantially all the yeast cells to grow on the xylose. The length oftime may vary depending on the population of genetically diversenon-recombinant yeast cells, and will also vary depending on the typesof medium used and how many cycles or repeats of steps (b) and (c) havebeen conducted. As each cycle is repeated, it is envisaged that the timewill become less as the method screens or selects for populations thatgrow faster using xylose with each repeat of steps (b) and (c),independent of whether solid or liquid medium is used. It will also beappreciated by persons skilled in the art that even in complex mediumcontaining sugars other than xylose, it is envisaged that some or all ofthe sugars other than xylose will eventually be depleted through growth,and that the presence of xylose will therefore eventually confer aselective advantage to those strains that grow faster on xylose as asole carbon source.

The yeast cells may be incubated in liquid xylose-containing medium fora sufficient amount of time to permit the yeast cells that are mostefficient at growing on xylose as a sole carbon source to outgrow thoseyeast cells that are less efficient at growing on xylose. Typically theamount of time is sufficient to permit growth of even those yeast cellsthat do not have an increased growth rate using xylose as a sole carbonsource. As discussed above, faster growing cells will be greater innumbers and therefore selected from. Thus, the use of liquidxylose-containing medium provides a convenient way to select for thoseyeast cells that are capable of utilising xylose at a faster rate thanthe rest of the yeast cell population. The period of growth in liquidxylose-containing medium is typically reduced with each cycle as thescreened or selected yeast cells become more efficient at utilisingxylose. Following screening or selecting of the yeast cells by culturingthe cells in liquid xylose-containing medium, the cells are typicallyharvested from the liquid medium and mated without further separation orisolation of the cells. Thus, the screened or selected yeast cells aretypically mated as a pool. The mating methods are the same as thosefollowing screening and selection on solid xylose-containing medium.

It will be appreciated that the yeast cells may be incubated in or onxylose-containing medium under any conditions which select or screen forxylose-utilising yeast strains in populations. By way of example,populations could be cultured:

-   -   (a) in or on xylose minimal medium under aerobic,        microaerophilic, anaerobic conditions;    -   (b) in or on xylose rich medium under aerobic conditions,        microaerophilic, anaerobic conditions.

The above medium may include other carbon sources (eg. sugars such asglucose, galactose, polyols such as xylitol, glycerol, organic acids andtheir salts such as acetic acid and acetates etc.) in addition toxylose. For example, the yeast cells may be exposed to stresses such assub- or supra-optimal pH, hyper- or hypo-osmotic pressure, ionicstresses from salts, alcohol stress from addition of ethanol or otheralcohols, stress from other organic inhibitors such as furfurals andtheir derivatives, sub- or supra-optimal temperatures, presence ofabsence of xylose and then subsequently selected or screened for theirability to recover from the stresses whilst utilising xylose as the solecarbon source. It is anticipated that combinations of differentselective conditions could be applied to the populations.

It will be appreciated that step (c) may be repeated any number of timesbefore isolating the yeast cells or repeating step (b). For example,populations selected in liquid xylose-containing medium may becontinually subcultured into fresh medium to further screen or selectfor yeast cells having the desired growth rate.

It will also be appreciated by those skilled in the art that steps (b)and (c) may be repeated any number of times to screen or select fornon-recombinant yeast cells in which the rate of growth on xylose as asole carbon source is progressively increased with each repeated cycle.This process is thus repeated for as many times as is required in orderto obtain a strain that exhibits the desired growth rate using xylose asa sole carbon source.

Thereafter, the method typically comprises the step of isolating one ormore yeast cells which have the desired growth rate. It will beappreciated by persons skilled in the art that the method typicallygenerates a population of genetically diverse non-recombinant yeastcells of Saccharomyces which have the desired growth rate.

Accordingly, in one embodiment, the method may be used to produce apopulation of Saccharomyces strains which are capable of growthutilising xylose as a sole carbon source at a desired growth rate. Thepopulation may be isolated without separating individual strains. Thismay be achieved, for example, by simply pooling the population ofSaccharomyces strains that are produced by the method.

In another embodiment, individual strains of Saccharomyces which arecapable of growth utilising xylose as a sole carbon source may beisolated. This may be achieved using standard microbiological techniquessuch as providing suitable colonies, streaking the yeast on agar platesfor single colony isolates, or any other methods for isolation of purecultures. Such methods are described in Cruickshank et al. (1975)“Medical Microbiology”, 12^(th) edition, volume two: The practice ofmedical microbiology, published by Churchill Livingstone.

Also provided is a method of generating a derivative of a Saccharomycesstrain with increased growth rate on xylose as a sole carbon source forgrowth. The Saccharomyces strain from which the derivative is generatedtypically has a desired property. The method comprises step (a) ofproviding the strain as part of a population of genetically diversenon-recombinant yeast cells of Saccharomyces. In other words, yeastcells of the strain make up a portion of the population. The yeast cellsof the genetically diverse population may be from any of the sourcesmentioned above. In step (b), the yeast cells of the population ofgenetically diverse yeast cells are cultured under any of the conditionsmentioned above which permit mating of the yeast cells of thepopulation. In step (c), the yeast cells are screened or selected forderivatives which have an increased growth rate. The increased growthrate is an increase in growth rate of the derivative relative to thegrowth rate of the strain. Typically, the yeast cells are screened orselected by incubating the yeast cell in or on xylose containing mediumas mentioned above. The xylose-containing medium may in addition containfactors which select or screen for the derivative strain. For example,the strain may comprise selectable markers such as antibiotic resistancemarkers or other types of markers which permit derivatives of the strainto be distinguished from other yeast cells of the population ofgenetically diverse yeast cells. This allows simultaneous screening orselection for yeast cells having increased growth using xylose as a solecarbon source, and carrying the selectable marker. This permitsdistinguishing the derivatives from the rest of the genetically diversepopulation. Suitable selectable markers include, for example, ADE2,HIS3, LEU2, URA3, LYS2, MET15, TRP1, URA4, sulfite resistance orp-fluoro-DL-phenylalanine resistance (Cebollero and Gonzalez (2004)Applied and Environmental Microbiology, Vol 70: 7018-7028). Methods forscreening and selecting for growth using xylose are as mentioned above.Steps (b) and (c) may be repeated any number of times until a derivativeis obtained with an increase in growth rate. Once the growth rate hasbeen increased, the derivative is isolated. Methods for isolated are asmentioned above.

Also included within the scope of the present invention areSaccharomyces strains produced by the method of the invention. TheSaccharomyces strain may be any species of Saccharomyces as definedphylogenetically by Kurtzman (2003) FEMS Yeast Research 3:417-432, andinclude S. cerevisiae, S. paradoxus, S. mikatae, S. cariocanus, S.kudriavzevii, S. pastorianus and S. bayanus. Methods for mating betweenstrains of Saccharomyces cerevisiae and non-cerevisiae strains arediscussed in, for example, Johnston J R and Oberman H (1979) YeastGenetics in Industry, in Bull M J (ed.) Progress in IndustrialMicrobiology, Elsevier, Amsterdam vol 15, pp. 151-205; Pretorius I S(2000) Tailoring wine yeast for the new millenium: novel approaches tothe ancient art of wine making. Yeast 16: 675-729; P. V. Attfield and P.J. L. Bell (2003) Genetics and classical genetic manipulations ofindustrial yeasts, in Topics in Current Genetics Vol. 2, J. H. de Winde(ed) Functional Genetics of Industrial Yeasts, Springer-Verlag BerlinHeidelberg.

It will be appreciated by persons skilled in the art that once a strainof Saccharomyces is obtained that is capable of utilising xylose as asole carbon source for growth at a desired growth rate, strains may bederived from that strain using methods known in the art for strainproduction including for example, classical genetic crossing methods,mutagenesis methods, recombinant methods, or any other methods forgenerating strains of Saccharomyces.

The strain capable of a growth rate of at least one generation per 48hours utilising xylose as a sole carbon source may be mated with otherstrains of Saccharomyces, preferably with other strains of Saccharomycescerevisiae. For example, it is envisaged that the above methods mayproduce multiple strains of Saccharomyces that are capable of a growthrate of at least one generation per 48 hours utilising xylose as a solecarbon source for growth. Thus, a first strain capable of a growth rateof at least one generation per 48 hours utilising xylose as a solecarbon source for growth may be mated with a second strain capable of agrowth rate of at least one generation per 48 hours utilising xylose asa sole carbon source for growth. Matings of this type may be performed,for example, to obtain a strain of Saccharomyces with even furtherenhanced or improved ability to utilise xylose as a sole carbon source.For example, it is envisaged that different strains of Saccharomycesthat are capable of rapid growth utilising xylose as a sole carbonsource may be mated to obtain mated strains that are capable of fastergrowth on xylose, or that are more efficient at producing products suchas ethanol or carbon dioxide when using xylose as a carbon source.

Strains of Saccharomyces that are capable of growth rates of at leastone generation per 48 hours using xylose as a sole carbon source may bemated with strains of Saccharomyces that are less capable of utilisingxylose as a sole carbon source. Matings of this type may be performed,for example, to transfer the improved ability to utilise xylose as asole carbon source to a Saccharomyces strain that has one or moredesirable properties not found in the strain that has an improvedability to utilise xylose as a sole carbon source. For example, aSaccharomyces cerevisiae strain less capable of using xylose as a solecarbon source for growth may have desirable characteristics for thebaking industry, and it may be advantageous to mate this strain with astrain that is capable of growth, at a rate of at least one generationper 48 hours utilising xylose as a sole carbon source in order todevelop a baker's yeast that can be grown more rapidly or efficiently onxylose and used subsequently in baking applications. Similarly, yeaststhat can be used for other industrial purposes such as distilling, winemaking, yeast extracts, enzymes, heterologous proteins, or any otherpurposes may be mated with strains which have an improved ability to usexylose as a sole carbon source in order to enable production ofbiomasses or yeast by-products on xylose media.

By mating a Saccharomyces strain, for example, a Saccharomycescerevisiae strain, in which the capability to grow at a rate of at leastone generation per 48 hours utilising xylose as a sole carbon source isobtained by non-recombinant methods with a recombinant strain in whichone or more genes for xylose utilisation have been introduced byrecombinant methods, it is envisaged that strains with even furtherimprovements in xylose utilisation can be produced. It will beappreciated by those skilled in the art that the genes for xyloseutilisation that have been introduced recombinantly would simplysupplement those genes already present in the Strain throughnon-recombinant methods.

It will also be appreciated by persons skilled in the art that while theability to utilise xylose is obtained by non-recombinant methods,recombinant DNA technology may be used to supplement the capability ofthe strain to utilise xylose as a sole carbon source. For example, oneor more xylose utilisation genes from other sources may be introducedinto the strain to supplement xylose utilisation. For example, xyloseisomerase from Piromyces sp. may be integrated into the genome asdescribed in Kuyper et al. merely to supplement xylose utilisation.Xylose reductase (XYL 1), or xylitol dehydrogenase (XYL2) from Pichiastipitis may be cloned into Saccharomyces as described in Wahlbom et al.(2003) to supplement the capability of the strain to utilise xylose.However, it will be understood by persons skilled in the art that theaddition of sequences for utilisation of xylose by recombinant methodsis merely to supplement the strains existing capability of a growth rateof at least one generation per 48 hours utilising xylose as a solecarbon source.

In a further example, one or more genes that encode for metabolicactivities that might impinge on efficiency of xylose utilisation bySaccharomyces other than through direct actions on xylose, xylitol orxylulose, could be introduced into the non-recombinant strains and theirexpression modified using recombinant DNA techniques. In some cases itmight be desirable to increase the expression of certain genes whereasin other cases it might be desirable to reduce or eliminate expressionof certain genes. Target genes for altered expression could includethose encoding cytoplasmic, mitochondrial or other organelle metabolicactivities. Such genes could encode for activities involved in sugartransport, nutrients transport, glycolysis, terminal steps offermentation, pentose phosphate pathway, gluconeogenesis, tricarboxylicacid pathway, glyoxylate cycle, electron transport chain, intracellularredox balance, interaction between fermentation and respiration, aminoacid synthesis and metabolism etc. Examples of genes that might bemodified by recombinant DNA technologies include RPE1, RK11, TAL1, andTKL1 or any other genes that might improve the ability of yeasts to usexylose as a sole carbon source.

It will be further appreciated by a person skilled in the art that oneor more genes for xylose utilisation may be introduced into the strainusing recombinant methods. Methods for the production of recombinantyeast are well known in the art and are described in for example,Guthrie and Fink (1991) “Guide to Yeast Genetics and Molecular Biology”,Methods in Enzymology Vol 194, Academic Press. Genes or sequences ofinterest may be cloned into suitable vectors for transformation into theyeast cell. The gene or sequence of interest is cloned into a suitableexpression vector such as pMA91 Dobson et al. (1984) EMBO Journal 3:1115), which comprises the appropriate regulatory regions for expressionin the yeast strain. Other vectors include episomal, centromeric,integrative, modified expression vectors known to those skilled in theart (see for example, Guthrie and Fink (1991) “Guide to Yeast Geneticsand Molecular Biology”, Methods in Enzymology Vol 194, Academic Press).Regulatory regions that may be suitable for expression in yeast mayinclude, for example, MAL, PGK1, ADH1, GAL1, GAL10, CUP1, GAP, CYC1,PHO5. Alternatively, the regulatory regions of the gene itself may beused to express the gene in Saccharomyces. The gene or sequence ofinterest may be integrated into the yeast genome, such as into theribosomal RNA locus, for instance. For this purpose, the ribosomalsequences of a suitable vector (eg. plasmid pIRL9) are released, andcloned appropriately to a BS+ vector. The gene or sequence of interestis operably linked to suitable yeast promoter and terminator regions toform an expression cassette, and the expression cassette is subsequentlycloned into the cloned ribosomal sequences. From this resulting plasmidthe expression cassette, flanked by ribosomal sequences can be releasedas a single fragment using appropriate restriction enzymes. The releasedfragment can be cotransformed with an autonomously replicating plasmidcarrying a suitable marker for transformation into a yeast using methodsknown in the art (see for example Guthrie and Fink (1991) “Guide toYeast Genetics and Molecular Biology”, Methods in Enzymology Vol 194,Academic Press). The plasmid can be later removed from the cell bycultivating the cells in conditions that do not select for the plasmid.

The Saccharomyces spp. strain of the present invention may be used forany use for which a Saccharomyces spp. strain of that species would beused. For example, a strain of Saccharomyces cerevisiae that is capableof utilising xylose as a sole carbon source may be used in baking,brewing, biomass production, sugar fermentation and ethanol production,or any other uses to which standard Saccharomyces strain are used.

The ability of the Saccharomyces strain of the present invention to growon xylose as a sole carbon source provides a convenient phenotype thatcan be used to distinguish between Saccharomyces strain which arecapable of growth rates of at least one generation per 48 hours usingxylose as a sole carbon source, and those that are not for purposes of“marking” yeast strains.

In one aspect, there is provided a method of marking a Saccharomycesstrain, comprising the steps of:

-   -   (a) culturing the desired strain with a strain of the invention        under conditions to permit combining of DNA of the strains;    -   (b) selecting or screening for derivatives of the desired strain        which have an increased growth rate using xylose as a sole        carbon source for growth;    -   (c) isolating derivatives of the desired strain which have an        increased growth rate using xylose as a sole carbon source for        growth.

In one embodiment, the method comprises:

-   -   (a) mating the desired strain with a Saccharomyces strain of the        fourth to fourteenth aspect under conditions which permit        combining of DNA of the strains; and    -   (b) screening or selecting for a derivative of the desired        strain that is capable of growing at a rate of greater than one        generation per 48 hours using xylose as a sole carbon source by        plating the mated cells on xylose-containing solid medium or by        culturing in xylose-containing liquid medium or a combination of        both;    -   (c) isolating derivatives of the desired strain which have an        increased growth rate using xylose as a sole carbon source for        growth.

Marked strains may be detected by incubating the strain on or inxylose-containing minimal media and determining the growth rate, orcomparing the growth rate to that of a standard strain with known growthrate on or in xylose-containing medium. A suitable test air detecting amarked strain is Test T1, whereby a marked strain exhibits at least a2-fold increase in biomass in Test T1.

In another embodiment, the Saccharomyces spp. strain of the inventionmay be used for the production of compounds such as ethanol, xylitol,acetic acid, other yeast byproducts, enzymes, etc. Methods for theproduction of compounds may comprise the following steps:

-   -   (a) cultivating the Saccharomyces strain in a xylose-containing        medium under conditions which permit the strain to produce the        compound; and    -   (b) recovering the compound(s) that is produced by the strain.

The compounds may be one or more compounds or mixtures thereof that areproduced by Saccharomyces when utilising xylose as a carbon source. Forexample, the compound may be ethanol, xylitol, acetic acid, carbondioxide, or any component, metabolites and byproducts of yeast cells andtheir metabolism. Components of yeast cells may include enzymes,co-factors, vitamins, amino acids, peptides, proteins, nucleosides,nucleotides, oligonucleotides, DNA, RNA, cell wall components, glucans,mannoproteins, membrane components, lipids, sterols, storagecarbohydrates, trehalose, glycogen, organic acids, succinic acid, aceticacid, lactic acid, polyols such as glycerol, xylitol.

It will be appreciated by persons skilled in the art that the type ofcompounds produced by the Saccharomyces spp. strain may depend on thegrowth conditions to which the strain is subjected. For example,temperature, aerobic or anaerobic growth, pH of the medium, nitrogensource, presence of carbon sources other than xylose, other compounds inthe medium on their own or in combination may impact on the types ofcompounds that are produced by the Saccharomyces cerevisiae strain.However, precise conditions for production of any one or more compoundscan be readily determined by a person skilled in the art throughstandard procedures.

It will also be appreciated by persons skilled in the art that theSaccharomyces strains of the invention will be capable of utilisingsugars such as glucose, fructose, mannose, galactose, maltotriose,maltose, sucrose, melibiose, raffinose, xylose, melizitose,alpha-methyl-glucoside, trehalose, isomaltose. The strains may also becapable of using non-sugar carbon sources such as ethanol, acetate,glycerol, xylitol.

The strain of the invention will typically be capable of fermentingsugars such as glucose, fructose, mannose, galactose, maltotriose,maltose, sucrose, melibiose, raffinose, xylose, melizitose,alpha-methyl-glucoside, trehalose or isomaltose in addition to xylosefor production of carbon dioxide and ethanol.

The xylose in the xylose-containing medium may be in any form that canbe utilised by the yeast. For example, the xylose may be purified, or itmay be in a crude form such as a hemicellulose hydrolysate obtained fromacid hydrolysis of biomass such as sugar cane, straw, corn stover,timber products etc.

In the production of biomass and compounds, the inoculatedxylose-containing media is typically cultivated at a temperature rangeof between 22° C. and 40° C. for between 2 hours and 4 days.

The inventors have further found that in order to produce ethanol fromxylose using the strains of the invention, it is not necessary that thestrains exhibit substantial growth on the xylose. The inventors havefound that heavy inocula of xylose-containing medium with strains of theinvention results in ethanol production within 2 hours, typically 4hours, more typically 5 hours. Under such conditions, substantial growthis not detectable. Thus, there is also provided a method of producingethanol comprising:

-   -   (a) inoculating xylose-containing media with the strain of the        first to eighth, twelfth or thirteenth aspects to a density of        at least 1×10⁸ yeast cells per ml of media;    -   (b) incubating the inoculated media for sufficient time to        permit ethanol production;    -   (c) recovering the ethanol.

The xylose containing media may be any of the xylose-containing mediamentioned above.

The density of yeast cells may be at least 5×10⁸, typically at least6×10⁸ yeast cells per ml of media.

The inoculated media may be incubated for at least 2 hours, typically atleast 5 hours.

In another aspect, there is provided a method of producing ethanolcomprising incubating a non-recombinant Saccharomyces strain inxylose-containing media under conditions sufficient to produce ethanoland thereafter recovering the ethanol.

Definition of Tests T1 to T9.

T1: Growth Using Xylose as Sole Carbon Source Yeast strains are streakedonto on Glucose Yeast extract Bacteriological Peptone medium solidifiedwith 2% Agar using standard microbiological techniques. After incubationfor 72 hours at 30 deg Celsius, yeast cells are taken from plates usinga sterile microbiological loop and inoculated to an OD₆₀₀ (OpticalDensity at 600 nm) of between 0.1 and 0.2 units (OD₆₀₀ at T₀) in 50 mlof broth. The broth contains xylose (5% w/v), Difco Yeast Nitrogen Basew/o amino acids (0.67%) in distilled water in a 250 ml Erlenmeyer flask.Cultures are incubated at 30 deg Celsius with shaking at 220 rpm (10 cmorbital diameter) for 48 hours prior to measuring OD₆₀₀ (OD atT_(48hrs)). The fold increase in biomass is determined by the equation:

$\frac{{OD}_{600}\mspace{14mu}{at}\mspace{14mu} T_{48\mspace{11mu}{hrs}}}{{OD}_{600}\mspace{14mu}{at}\mspace{14mu} T_{0}}.$T2: Cell Biomass Yield Using Xylose as Sole Carbon Source.

Yeast strains are streaked onto on Glucose Yeast extract BacteriologicalPeptone medium solidified with 2% Agar using standard microbiologicaltechniques. After incubation for 72 hours at 30 deg Celsius, yeast cellsare taken from plates using a sterile microbiological loop andinoculated to an OD₆₀₀ (Optical Density at 600 nm) of between 0.1 and0.2 units in 50 ml of broth. The broth contains xylose (5% w/v), DifcoYeast Nitrogen Base w/o amino acids (0.67%) in distilled water in a 250ml Erlenmeyer flask. Cultures are incubated at 30 deg Celsius withshaking at 220 rpm (10 cm orbital diameter) for 72 hours prior tomeasuring the yield of dry yeast matter. The dry weight content of theyeast is measured by transferring 5 mls of yeast culture to apre-weighed glass test tube (W1), followed by centrifugation at 3000 gfor 10 minutes at 22 deg C. The supernatant is removed withoutdisturbing the yeast pellet, and the cells are resuspended in 5 ml ofdistilled water, prior to re-centrifugation at 3000 g for 10 minutes at22 deg C. The supernatant is again removed without disturbing the yeastpellet, and the cells are resuspended in 5 ml of distilled water, priorto re-centrifugation at 3000 g for 10 minutes at 22 deg C. The glasstest tube containing the yeast cells is baked at 105 deg C. for 24 hoursand weighed (W2). Dry yeast matter is calculated by subtracting W1 fromW2 and multiplying the obtained value by 10. Assays are performed induplicate and the average is calculated.

T3: Production of Ethanol Using Xylose as Sole Carbon Source.

Inoculum of yeast was prepared by growing 1 standard microbial loopfulof pure cells of the strain for 16 hr at 30° C. with shaking at 200 rpmin a 250 mL Erlenmeyer flask containing in 50 mL distilled water: 2.5 gxylose, 0.5 g yeast extract, 0.5 g bacteriological peptone. Seven×50 mLcultures were grown simultaneously. The cultures were harvested bycentrifugation at 22° C. and 3,000×g for 5 min. Supernatant wasdiscarded and cell pellets were resuspend in sterile distilled water andre-centrifuged. Supernatant was discarded and cell pellets resuspendedand pooled in 20 mL of xylose minimal medium which contained per L ofdistilled water:

50 g xylose, 13.4 g Difco Yeast Nitrogen Base without amino acids,supplemented with 0.4 mg of CuSO₄.5H₂O, 1 mg ZnSO₄.7H₂O, 2 mg ofMnSO₄.4H₂O, 1 mg Na₂MoO₄.2H₂O, 1 mg of Na₂B₄O₇.10H₂O, 2 mgCa-pantothenate, 2 mg thiamine HCl, 2 mg pyridoxine HCl, 4 mg inositol,1 mg nicotinic acid, and 0.4 mg biotin.

The cells were then inoculated into 980 mL of the same xylose minimalmedium which had been prewarmed to 30° C. and aerated to 20% oxygen is aBraun Biostat B 2 L fermentation vessel. Yeast was grown with air pumpedat 10 L per min, stirring at 1200 rpm and pH maintained at pH5 usingadditions of KOH or phosphoric acid as required. After 24 h, 300 mL ofthe culture volume was removed and replaced with 300 ml fresh xyloseminimal medium comprising 50 g xylose plus 10 g Difco Yeast NitrogenBase without amino acids and trace salts and vitamins in the amountsdescribed above. After a further 7 h 30 g of fresh xylose was added tothe culture and air supply was reduced to 4 L/min and stirrer speedreduced to 200 rpm. Ethanol was assayed after a further 20 h by use of aYSI 2700 Select Biochemistry Analyzer fitted with a YSI membrane 2786for ethanol detection (YSI Inc. Yellow Springs, Ohio, USA).

T4: Assay of Xylose reductase:

Strains of yeast were grown on glucose, yeast extract andbacteriological peptone agar as described previously, were inoculated toan optical density at 600 nm of between 0.1 and 0.2 units in a 250 mLshake flask in 50 mL medium that contained 5% w/v xylose, 0.5% w/v yeastextract and 1% w/v bacteriological peptone (XYP). Cultures wereincubated at 30° C. and 180 rpm until they reached optical density at600 nm of between 3 and 5 units. If cells did not reach the requireddensity after 24 h incubation they were nevertheless harvested. Cellswere harvested by centrifugation at 3,000×g and 4° C. for 5 min. Thesupernatant was discarded and the cell pellet was resuspended in chilleddistilled water and re-centrifuged. Supernatant was discarded and theprocess repeated so as to remove all traces of medium.

Cells were re-suspended in disintegration buffer and cell extractsprepared as described in Eliasson A., et al. (2000) Applied andEnvironmental Microbiology, volume 66 pages 3381-3386. The cell extractswere then assayed for xylose reductase activities according to themethods described and referred to in Eliasson A., et al. (2000) Appliedand Environmental Microbiology, volume 66 pages 3381-3386. One unit ofactivity is defined as 1 nanomole of NAD(P)H reduced or oxidised perminute per mg of protein at 30° C. Protein was assayed by the methoddescribed by Lowry O H, Rosebrough N J, Farr A L, and Randall R J (1951)Journal of Biological Chemistry 193:265-275, using a bovine serumalbumin standard.

T5: Assay of Xylitol dehydrogenase.

Strains of yeast were grown on glucose, yeast extract andbacteriological peptone agar as described previously, were inoculated toan optical density at 600 nm of between 0.1 and 0.2 units in a 250 mLshake flask in 50 mL medium that contained 5% w/v xylose, 0.5% w/v yeastextract and 1% w/v bacteriological peptone (XYP). Cultures wereincubated at 30° C. and 180 rpm until they reached optical density at600 nm of between 3 and 5 units. If cells did not reach the requireddensity after 24 h incubation they were nevertheless harvested. Cellswere harvested by centrifugation at 3,000×g and 4° C. for 5 min. Thesupernatant was discarded and the cell pellet was resuspended in chilleddistilled water and re-centrifuged. Supernatant was discarded and theprocess repeated so as to remove all traces of medium.

Cells were re-suspended in disintegration buffer and cell extractsprepared as described in Eliasson A., et al. (2000) Applied andEnvironmental Microbiology, volume 66 pages 3381-3386. The cell extractswere then assayed for xylitol dehydrogenase activities according to themethods described and referred to in Eliasson A., et al. (2000) Appliedand Environmental Microbiology, volume 66 pages 3381-3386. One unit ofactivity is defined as 1 nanomole of NAD(P)H reduced or oxidised perminute per mg of protein at 30° C. Protein was assayed by the methoddescribed by Lowry O H, Rosebrough N J, Farr A L, and Randall R J (1951)Journal of Biological Chemistry 193:265-275, using a bovine serumalbumin standard.

T6: Assay of Xylulose kinase.

Strains of yeast were grown on glucose, yeast extract andbacteriological peptone agar as described previously, were inoculated toan optical density at 600 nm of between 0.1 and 0.2 units in a 250 mLshake flask in 50 mL medium that contained 5% w/v xylose, 0.5% w/v yeastextract and 1% w/v bacteriological peptone (XYP). Cultures wereincubated at 30° C. and 180 rpm until they reached optical density at600 nm of between 3 and 5 units. If cells did not reach the requireddensity after 24 h incubation they were nevertheless harvested. Cellswere harvested by centrifugation at 3,000×g and 4° C. for 5 min. Thesupernatant was discarded and the cell pellet was resuspended in chilleddistilled water and re-centrifuged. Supernatant was discarded and theprocess repeated so as to remove all traces of medium.

Cells were re-suspended in disintegration buffer and cell extractsprepared as described in Eliasson A., et al. (2000) Applied andEnvironmental Microbiology, volume 66 pages 3381-3386. The cell extractswere then assayed for xylulose kinase activities according to themethods described and referred to in Eliasson A., et al. (2000) Appliedand Environmental Microbiology, volume 66 pages 3381-3386. One unit ofactivity is defined as 1 nanomole of NAD(P)H reduced or oxidised perminute per mg of protein at 30° C. Protein was assayed by the methoddescribed by Lowry O H, Rosebrough N J, Farr A L, and Randall R J (1951)Journal of Biological Chemistry 193:265-275, using a bovine serumalbumin standard.

T7: Growth Using Xylitol as Sole Carbon Source

Yeast strains are streaked onto on Glucose Yeast extract BacteriologicalPeptone medium solidified with 2% Agar using standard microbiologicaltechniques. After incubation for 72 hours at 30 deg Celsius, yeast cellsare taken from plates using a sterile microbiological loop andinoculated to an OD₆₀₀ (Optical Density at 600 nm) of between 0.1 and0.2 units (OD₆₀₀ at T_(o)) in 50 ml of broth. The broth contains xylitol(5% w/v), Difco Yeast Nitrogen Base w/o amino acids (0.67%) in distilledwater in a 250 ml Erlenmeyer flask. Cultures are incubated at 30 degCelsius with shaking at 220 rpm (10 cm orbital diameter) for 48 hoursprior to measuring OD₆₀₀ (OD₆₀₀ at T_(48hrs)). The fold increase inbiomass is determined by the equation:

$\frac{{OD}_{600}\mspace{14mu}{at}\mspace{14mu} T_{48\mspace{11mu}{hrs}}}{{OD}_{600}\mspace{14mu}{at}\mspace{14mu} T_{0}}$T8: Ethanol Production Using Xylose in a Rich Medium.

Yeast strains are streaked onto on Glucose, Yeast Extract,Bacteriological Peptone, medium solidified with 2% Agar using standardmicrobiological techniques. After incubation for 96 hours at 30 degCelsius, yeast cells are taken from plates using a sterilemicrobiological loop and inoculated to an OD₆₀₀ (Optical Density at 600nm) of between 1 and 2 units in 50 ml of broth. The broth containsxylose (5% w/v), Yeast extract (0.5% w/v) and Bacteriological Peptone(1% w/v) in distilled water in a 250 ml Erlenmeyer flask. Cultures areincubated at 30 deg Celsius with shaking at 220 rpm (10 cm orbitaldiameter). Samples of the culture supernatant are assayed hourly for aperiod of 24 hours for ethanol by use of a YSI 2700 Select BiochemistryAnalyzer fitted with a YSI membrane 2786 for ethanol detection (YSI Inc.Yellow Springs, Ohio, USA). Levels of ethanol are expressed as grams perliter.

T9: Ethanol Production in Xylose Minimal Mineral Medium Under AnaerobicConditions.

Yeast strains were streaked onto on 5% w/v xylose, 0.67% w/v Difco YeastNitrogen Base without amino acids medium solidified with 2% agar usingstandard microbiological techniques. After incubation for 96 hours at 30deg Celsius, yeast cells are taken from plates and inoculated directlyinto 10 mL of sterile 5% w/v xylose plus 0.67% w/v Difco Yeast NitrogenBase without amino acids contained in a 15 mL volume sterile PP-TestTubes (Cellstar, Greiner bio-one) to an optical density at 600 nm of 0.1to 0.4. Medium was overlayed with 2 mL sterile mineral oil to inhibitoxygen transfer, screw caps fully tightened and tubes were incubatedwithout shaking at 30° C. Samples for ethanol assay we removed usingsterile Pasteur pipettes, without disturbing cell pellets that hadformed through growth of original inoculum. Ethanol was assayed using aYSI 2700 Select Biochemistry Analyzer fitted with a YSI membrane 2786for ethanol detection (YSI Inc. Yellow Springs, Ohio, USA).

The invention will now be described in detail by way of reference onlyto the following non-limiting examples.

EXAMPLE 1

Saccharomyces is Capable of Slow Growth Using Xylose as the Sole CarbonSource

Baker's yeast Saccharomyces cerevisiae strain NL67 (Higgins et al.(1999) Applied and Environmental Microbiology 65:680-685) was inoculatedonto solidified minimal mineral medium with or without xylose as a solecarbon source and incubated at 30° C. for two months. It was observedusing a light microscope that microscopic colonies occurred on bothtypes of plates, but that the colonies on the medium containing xylosewere detectably larger than those provided with no carbon source.Whereas the cells on medium containing no xylose had progressed through5 to 6 generations, the cells on medium containing xylose had progressedthrough 9 to 10 generations.

EXAMPLE 2

Generation of Populations Comprising Diverse Strains of SaccharomycesCapable of Rapid Growth on Xylose as Sole Carbon Source

By obtaining yeast strains from a variety of sources, which includedstrains obtained from the wild, from wine, distiller's and beerfermentations, and baking applications, inducing them to sporulate andusing mass mating techniques to generate a genetically diversepopulation it was possible to apply selection pressure to enrich foryeast strains capable of more vigorous growth using xylose as a solecarbon source. By spreading the genetically diverse populations ontoxylose minimal mineral medium (and simultaneously onto the same minimalmineral medium without a carbon source) and incubating for two months,it was observed that there was heterogeneity in the colony size. Bycomparing the plates with xylose and without xylose, the observation wasmade that contrary to accepted dogma, growth had been due to theaddition of xylose to the medium; implying that there was heterogeneityin the ability of the yeast strains within the population to grow onxylose. By harvesting the entire xylose grown population, andsporulating them, it was possible to generate new populations of yeastthat were enriched for genetic information conferring ability to growmore effectively on xylose. By using large population sizes (at least100,000) and pooling cells including those that did not grow optimally,the genetic diversity of the population was maintained in eachgeneration. As the number of cycles of mating and selection increased,the heterogeneity of colony sizes was maintained but the final size ofthe colonies increased.

After between 5 and twenty cycles of selection on agar plates, thepopulations were introduced into liquid culture containing minimalmineral medium with xylose as a sole carbon source, and continuallysub-cultured for approximately 50 generations. The subsequentpopulations were sporulated and new populations were constructed bymass-mating. The new populations of heterogeneous Saccharomyces cellswere grown in liquid culture in xylose minimal medium for approximately50 generations. After this time some of the sample was stored and someof the sample was sporulated. Germinated spores derived from theselected population were mated en masse to generate a new population ofheterogeneous Saccharomyces cerevisiae cells to grow under selectionpressure in liquid xylose minimal medium for approx. 50 generations.This process can be reiterated until desired growth rates are achieved.

To determine whether growth rates of populations on xylose minimalmedium were increasing, samples taken after 365, 569, 1013, 1059, 1170,and 1377 days respectively of selection were inoculated into xyloseminimal medium at an optical density of 600 nm (OD) between 0.1 and 0.2with shaking at 220 rpm and 30 deg C. OD was re-assayed after 24 h andused to calculate average doubling time over the 24 h period accordingto standard microbiological method. The results were plotted graphicallyand are shown in FIG. 1.

FIG. 1 shows the improvement in growth rates of the populations en masseis exponential as they progress through rounds of mating and selectionfor growth on xylose as the sole carbon source. This improvement ingrowth rate on xylose as sole carbon source is predicted to continueuntil growth rate on xylose is equivalent to the growth rate on glucoseas sole carbon source.

EXAMPLE 3

Ethanol Production in Xylose Rich Medium by Heterogeneous Populations ofNon-Recombinant Saccharomyces Strains Correlates with Growth Rate onXylose Minimal Mineral Medium

Doubling times of the populations in xylose minimal medium weredetermined as described in Example 2, FIG. 1. Ethanol production wasdetermined by inoculating samples of populations at an optical densityat 600 nm of between 1 and 2 into 250 mL shake flasks at in 50 mL mediumthat contained 5% w/v xylose, 0.5% w/v yeast extract and 1% w/vbacteriological peptone (XYP). Cultures were incubated at 30° C. and 220rpm and ethanol production monitored over a 2 day period.

The data obtained (Table 1) indicate that the ability to produce ethanolin xylose rich medium increased as doubling time of heterogeneouspopulations on xylose minimal medium was reduced. Both characteristicsimproved in concert with the number of reiterations and length of timethat the mating and selection process was applied.

TABLE 1 Ethanolic fermentation of xylose in rich medium improves asgrowth rate of heterogeneous populations of non-recombinantSaccharomyces in xylose minimal mineral medium increases Doubling timeMinutes incubation in Xylose rich medium (h) in xylose 1035 1155 12751395 1455 2465 Population minimal medium min min min min min min 1 142 00 0 0 0 0 2 76 0 0 0 0 0 0 3 17 0 0 0 0 0 .02 4 14 0 0 0 0.01 0.01 0.025 10 0 0 0.06 0.09 0.12 0.69 6 7 0.01 0.05 0.14 0.16 0.21 1.1 Numbersfor ethanol production represent g ethanol/L produced at the time (min)indicated.

EXAMPLE 4

Characterization of Pure Non-Recombinant Strains of SaccharomycesCapable of Growth on Xylose as Sole Carbon Source According to Tests T1and T2

Saccharomyces strain isolates were purified from xylose-utilisingpopulations of Example 2 by standard microbiological procedure andtested for growth and yields on xylose as sole carbon source accordingto Test T1 and T2. Strains CEN.PK (Karhumaa et al. (2005) Yeast22:259-368) and NL67 (Higgins et al. (1999) Applied and EnvironmentalMicrobiology 65:680-685) were included as representing strain typesexisting prior to the application of the methods described herein.Strains NM04/41257 and NM04/41258 (deposited) are derived frompopulations that had gone through 1059 days of running of the protocoldescribed in Example 2 and FIG. 1. Strains ISO 10 (NM 05/45177) and ISO7 (NM 05/45178) were obtained from populations that had gone through1377 days, and 1431 days, respectively, of running of the protocoldescribed in Example 2 and FIG. 1. The individual isolated yeast strainswere confirmed to be members of the species Saccharomyces cerevisiae bysporulation and mating of subsequently derived haploids with knownlaboratory strains of Saccharomyces cerevisiae with genetic markers.

TABLE 2 Growth of pure strains of Saccharomyces cerevisiae on xyloseminimal medium as described in Test T1. Initial Optical Final OpticalFold STRAIN Density Density Increase CEN.PK 0.128 0.132 1.03 NL67 0.1540.198 1.29 NM04/41257 0.111 2.57 23.15 NM04/41258 0.110 2.9 26.36 ISO 100.127 6.3 49.61 NM 05/45177 ISO 7 0.170 6.8 40 NM 05/45178

Biomass yields of pure strains of Saccharomyces cerevisiae on xyloseminimal medium were assayed as described in Test T2. Strains CEN.PK andNL67 were again included as representing strain types that have not beengenerated using the methods described herein. CEN.PK yield=1 mg dryyeast matter per 50 mL culture; NL67 yield=2 mg dry yeast matter per 50mL culture; NM04/41257 yield=50 mg dry yeast matter per 50 mL culture;NM04/41258 yield=55 mg dry yeast matter per 50 mL culture; ISO 10yield=145 mg dry yeast matter per 50 mL culture, ISO 7 yield=114 mg dryyeast matter per 50 mL culture.

The above data indicate that the protocol generates strains that possessthe ability to rapidly grow and yield well using xylose as a sole carbonsource. Taken with the populations data in Example 2 and FIG. 1 the datademonstrate that the protocol can be applied reiteratively to derivestrains that have a maximum possible growth rate and yield on xylose,which could be equivalent to growth rate and yield on glucose.

EXAMPLE 5

Non-Recombinant Saccharomyces Strains Capable of Rapid Growth and ofProducing Ethanol Using Xylose as the Sole Carbon Source

Inoculum of strain ISO10 was prepared by growing 1 standard microbialloopful of pure cells of the strain for 16 hr at 30° C. with shaking at200 rpm in a 250 mL Erlenmeyer flask containing in 50 mL distilledwater: 2.5 g xylose, 0.5 g yeast extract, 0.5 g bacteriological peptone.Seven×50 mL cultures were grown simultaneously. The cultures wereharvested by centrifugation at 22° C. and 3,000×g for 5 min. Supernatantwas discarded and cell pellets were re-suspended in sterile distilledwater and re-centrifuged. Supernatant was discarded and cell pelletsre-suspended and pooled in 20 mL of xylose minimal medium whichcontained per L of distilled water: 50 g xylose, 13.4 g Difco YeastNitrogen Base without amino acids, supplemented with 0.4 mg ofCuSO₄.5H₂O, 1 mg ZnSO₄.7H₂O, 2 mg of MnSO₄.4H₂O, 1 mg Na₂MoO₄.2H₂O, 1 mgof Na₂B₄O₇.10H₂O, 2 mg Ca-pantothenate, 2 mg thiamine HCl, 2 mgpyridoxine HCl, 4 mg inositol, 1 mg nicotinic acid, and 0.4 mg biotin.

The cells were then inoculated into 980 mL of the same xylose minimalmedium which had been pre-warmed to 30° C. and aerated to 20% oxygen isa Braun Biostat B 2 L fermentation vessel. Yeast was grown with airpumped at 10 L per min, stirring at 1200 rpm and pH maintained at pH5using additions of KOH or phosphoric acid as required. After 24 h, 300mL of the culture volume was removed and replaced with 300 ml freshxylose minimal medium comprising 50 g xylose plus 10 g Difco YeastNitrogen Base without amino acids and trace salts and vitamins in theamounts described above. This gave a culture of cell mass of 4 g dryyeast equivalents per L. Aeration and stirring was maintained at 10L/min and 1200 rpm, respectively and pH maintained at pH5. Under theseconditions yeast biomass doubled in 4 hours (based on dry yeast matter)with a yield of 4 g dry yeast matter from 10 g consumed xylose.

When yeast grown by the procedure described above reached a density ofabout 12 g (dry yeast equivalent) per L, 30 g of fresh xylose was addedto the culture and air supply was reduced to 4 L/min and stirrer speedreduced to 200 rpm. Under these conditions where dissolved oxygen wasundetectable, 17 g of xylose was consumed and a further 1 g dry yeastmatter was produced along with 1.75 g ethanol/L and 1 g xylitol/L in 20h.

EXAMPLE 6

Activities of Xylose Reductase (XR), Xylitol Dehydrogenase (XDH) andXylulokinase (XK) in Exponentially Growing Non-Recombinant Yeast Strains

Strains of yeast were grown on glucose, yeast extract andbacteriological peptone agar as described in test T1, were inoculated toan optical density at 600 nm of between 0.1 and 0.2 units in a 250 mLshake flask in 50 mL, medium that contained 5% w/v xylose, 0.5% w/vyeast extract and 1% w/v bacteriological peptone (XYP). Cultures wereincubated at 30° C. and 180 rpm until they reached optical density at600 nm of between 3 and 5 units. If, as in the case of strains such asNL67 and CEN.PK, cells did not reach the required density after 24 hincubation they were nevertheless harvested and assayed. Cells wereharvested by centrifugation at 3,000×g and 4° C. for 5 min. Thesupernatant was discarded and the cell pellet was resuspended in chilleddistilled water and re-centrifuged. Supernatant was discarded and theprocess repeated so as to remove all traces of medium.

Cells were re-suspended in disintegration buffer and cell extractsprepared as described in Eliasson A., et al. (2000) Applied andEnvironmental Microbiology, volume 66 pages 3381-3386. The cell extractswere then assayed for XR, XDH and XK activities according to the methodsdescribed and referred to in Eliasson A., et al. (2000) Applied andEnvironmental Microbiology, volume 66 pages 3381-3386. The followingactivities were found where one unit of activity is defined as 1nanomole of NAD(P)H reduced or oxidised per minute per mg of protein at30° C.

TABLE 3 Units of enzyme activities of yeast strains Strain XR activityXDH Activity XK Activity CEN.PK 0.74 0.025 23 NL67 0.74 0.12 20.2NM04/41257 1.16 1.09 20.9 NM04/41258 1.61 1.13 22.5 ISO 10 3.81 8.0922.5 ISO 7 5.39 13.35 29.75

These data suggest that the reiterative mating and selection methodologyapplied to obtain the yeasts that grow on xylose as the sole carbonsource has resulted in improved XR and XDH activities that are criticalfor metabolism of xylose and xylitol.

It is likely that other enzymes and activities such as those involved inpentose phosphate pathway have also been naturally improved in ourstrains due to the selective pressures applied in the breeding andselection procedures. Those skilled in the art would realize that ourstrains could obviously be used as a basis for further improvement usingfurther selections and combinations of breeding, mutagenesis, protoplastfusion, cytoduction and/or recombinant DNA techniques that optimizeactivities of XR, XDH, XK, or introduce and optimize xylose isomerase,or other genetic changes required to improve xylose, xylitol andxylulose metabolism.

EXAMPLE 7

Characterization of Pure Non-Recombinant Strains of SaccharomycesCapable of Growth on Xylitol as Sole Carbon Source According to Test T7

Pure strains were grown on glucose, yeast extract and bacteriologicalpeptone agar as described in test T1. Colonies of the strain wereinoculated in to 50 mL of 5% w/v xylitol plus 0.67% Difco Yeast NitrogenBase without Amino Acids and tested for growth as described in Test T7.

TABLE 4 Growth of pure strains of Saccharomyces cerevisiae on xylitolminimal medium as described in Test T7. Initial Optical Final OpticalFold STRAIN Density Density Increase CEN.PK 0.146 0.130 0.89 NL67 0.1560.164 1.05 NM04/41257 0.130 0.171 1.32 NM04/41258 0.105 3.03 28.86 ISO10 0.108 0.115 1.065 ISO 7 0.198 6.96 35.15

These data suggest that the reiterative mating and selection methodologyapplied to obtain the yeasts that grow on xylose as the sole carbonsource has resulted in some strains that can also utilize xylitol as thesole carbon source.

EXAMPLE 8

Production of Ethanol Using Xylose Rich Medium by Pure Non-RecombinantStrains of Saccharomyces Capable of Growth on Xylose as Sole CarbonSource According to Test T8

Pure isolates of yeasts grown on glucose, yeast extract andbacteriological peptone agar as described in test T1, were inoculated toan optical density at 600 nm of between 1 and 2 units in a 250 mL shakeflask in 50 mL medium that contained 5% w/v xylose, 0.5% w/v yeastextract and 1% w/v bacteriological peptone (XYP) and assayed asdescribed in Test T8.

TABLE 5 Ethanol production using xylose rich medium Initial OpticalOptical Density Ethanol (g/L) STRAIN Density after 24 h after 24 hCEN.PK 1.16 1.9 0 NL67 1.29 1.8 0 NM04/41257 1.06 14.5 0.04 NM04/412581.17 17.2 0.07 ISO 10 1.18 20.0 0.66 ISO 7 1.15 21.2 1.32

These data show that the selection strategy based on growth of yeasts onxylose as the sole carbon source generates yeast strains that arecapable of producing ethanol from xylose. These data coupled with dataof Example 3 (Table 1), show that the ability to grow and yield cellbiomass using xylose as the sole carbon sugar increases in concert withincreasing ability to produce ethanol from xylose. Moreover strains suchas NM04/41258 and ISO 7 that can utilize xylitol and xylose are able toproduce ethanol. The finding that strains NM04/41258 and ISO 7 (NM05/45178) which can utilize xylitol and make ethanol from xylose goesagainst the teaching of U.S. Pat. No. 4,511,656, which states strainsshould be screened to isolate the specific colony or colonies which willutilize D-xylose but not xylitol.

EXAMPLE 9

Isolation and Characterisation of Pure Non-Recombinant Strains ofSaccharomyces Capable of Ethanol Production Using Xylose as Sole CarbonSource Under Anaerobic Conditions

Isolates were purified from xylose-utilising populations that hadundergone 1106 days of selection and were subjected to Test T9. Fiftyindividual isolates were tested and ethanol production assayed afterthree weeks to 4 months ranged from 0.24 g/L to 0.75 g/L. StrainsNM04/41257 and NM04/41258 were also included in these assays.

TABLE 6 Production of ethanol by pure yeast strains in xylose minimalmedium under anaerobic conditions. g/L Ethanol Strain Incubation PeriodProduced NM04/41257 4 months 0.82 NM04/41258 4 months 0.48 Isolate no. 21 month 0.70 Isolate no. 6 1 month 0.75 Isolate no. 21 1 month 0.70Isolate no. 23 1 month 0.73 Isolate no. 31 3 weeks 0.64 Isolate no. 36 3weeks 0.63 Isolate no. 37 3 weeks 0.65

These data indicate that it is possible to obtain non-recombinantSaccharomyces yeasts that are capable of fermenting xylose underanaerobic conditions to produce ethanol. The NM04/41257 and NM04/41258strains were purified from earlier selected populations relative to theother strains in the Table. This data shows that the reiterativeprotocol described herein results in increased efficiency of anaerobicethanol production by non-recombinant strains.

EXAMPLE 10

Rapid Ethanol Production by Non-Recombinant Saccharomyces Inoculated atHigh Density into Minimal Media Under Aerobic Conditions

Strain ISO 10 grown on glucose, yeast extract, bacteriological peptoneagar (as described above) was inoculated into 50 mL of 5% w/v xylose,0.5% w/v yeast extract and 1% w/v bacteriological peptone (XYP)contained in 250 mL Erlenmeyer flasks and incubated for 72 h at 30° C.and 220 rpm. Twenty flasks were incubated simultaneously. Cells wereharvested by centrifugation at 3,000×g and 22° C. for 10 min.Supernatant was discarded and cell pellet re-suspended in 10 mL sterilewater before re-centrifugation. The supernatant was again discarded andthe Cells re-suspended in distilled sterile water before a furthercentrifugation. Finally, cells were re-suspended in 20 mL steriledistilled water.

Fermentation medium was prepared, filter-sterilized as follows: YNBContained 0.67% Difco Yeast Nitrogen Base without amino acids; XYNB wasYNB plus 5% xylose; GXYNB was YNB plus 0.5% glucose and 4.5% xylose.Media were contained in 125 mL conical glass flasks. They wereinoculated with 3 mL of cell suspension and placed on a 220 rpm orbitalshaker at 30° C. and ethanol production read at hourly intervals asdescribed previously. Immediately after inoculation optical densities at600 nm of cultures were measured and it was determined that celldensities for cultures were at 5.7 to 6.1×10e8 per mL.

TABLE 7 Production of ethanol by high, density inoculum ofNon-recombinant Saccharomyces cells. Medium 1 h 2 h 3 h 4 h 5 h YNB 0.410.44 0.36 0.29 0.09 XYKB 0.75 1.1 1.67 2.1 2.9 GXYNB 1.69 2.2 2.41 3.23.8

Numbers refer to g ethanol per L in culture supernatant at the indicatedtime after inoculation. The small amount of ethanol produced by cellsincubated in YNB without a carbon source was derived most likely fromcarry over of endogenous storage sugars synthesized and accumulated bythe yeast during the 72 h inoculum preparation phase. Decline in theethanol concentration of YNB cultures after 3 h indicates that storagesugars were exhausted or becoming exhausted and ethanol was beingconsumed by cells and/or ethanol was evaporating.

These data show that it is possible for non-recombinant strains ofSaccharomyces to be obtained that can ferment xylose rapidly to produceethanol in minimal medium where xylose is the sole carbon source.Moreover, these strains are able to produce ethanol from xylose inmedium where fermentable hexose sugar such as glucose was also addedsince the ethanol produced by 4 h in GXYNB was in excess of thatexpected solely from the glucose present.

EXAMPLE 11

Non-Recombinant Saccharomyces Biomass Grown on Xylose as the Sole CarbonSource Exhibits Broad Industrially Useful Features

Those skilled in the art will know that yeasts have proven industrialutility in various fermentation applications (e.g. bread, and ethanol(potable and non-potable) production) and also as a source of aminoacids and protein, nucleotides (e.g. DNA and RNA), enzymes (e.g.invertase and phytase), antioxidants (e.g. glutathione) and othercellular components such as cell wall components (e.g. glucans). Theaforementioned properties are by way of example and are not meant to belimiting.

It would be useful if yeasts could be grown on media that contain xyloseand then subsequently used for the various industrial purposes.Therefore, by way of example strain ISO10 was tested for variousfeatures following growth on xylose.

Strain ISO10 was grown in per Litre: 50 g xylose, 13.4 g Difco YeastNitrogen Base without amino acids, supplemented with 0.4 mg ofCuSO₄.5H₂O, 1 mg ZnSO₄.7H₂O, 2 mg of MnSO₄.4H₂O, 1 mg Na₂MoO₄.2H₂O, 1 mgof Na₂B₄O₇.10H₂O, 2 mg Ca-pantothenate, 2 mg thiamine HCl, 2 mgpyridoxine HCl, 4 mg inositol, 1 mg nicotinic acid, and 0.4 mg biotin.Cells were stirred at 1200 rpm with air supply at 12 L per min andtemperature at 30° C. and pH maintained at pH5 using 1M KOH and 1Mphosphoric acid. When cell density was at A₆₀₀ 14, cells were harvestedby centrifugation at 3,000×g and 22° C. for 10 min. Cell pellet wasresuspended in distilled water and re-centrifuged. This washingprocedure was carried out three-times and the biomass placed on Whatmanfilter paper no. 1 to achieve a wet biomass of 22 to 25% solids.

The biomass was assayed for the following features by way ofdemonstrating that it is possible to grow non-recombinant Saccharomyceson xylose as sole carbon source to obtain yeast biomass with propertiesgenerally relevant to industrial applications.

Ethanolic fermentation power. Yeast was inoculated into a molasses-basedmedium to test for ethanolic fermentation power, defined as the abilityto produce ethanol from fermentable sugars such as sucrose, glucose andfructose. Sugar cane molasses was diluted in water and sterilized byheating to 121° C. for 5 min. When cooled to 22° C., the molasses wascentrifuged at 4,000×g at 22° C. for 10 min to remove solids. Thesupernatant was diluted to a final concentration of 18% w/w sucroseequivalents and supplemented with filter-sterilised Difco Yeast NitrogenBase at 0.67% w/v. Forty mL of this medium was inoculated with anequivalent of 6.8 mg dry yeast matter and incubated at 30° C. withoutshaking. Ethanol was assayed at 24 h intervals by use of a YSI 2700Select Biochemistry Analyzer fitted with a YSI membrane 2786 for ethanoldetection (YSI Inc. Yellow Springs, Ohio, USA). After 24 h the yeastproduced 16.8 g ethanol per L, after 1 week the yeast had produced 59 gethanol per L.

Leavening of bread dough. To test for leavening power, defined as theability to ferment sugars such as glucose, fructose and maltose andthereby produce carbon dioxide which raises a flour dough, the yeast wasadded to a bread dough mixture and tested for its ability to produceleavened bread. A dough was prepared that contained 500 g wholemealflour soy and linseed bread mix (Kitchen Collection, Christchurch), 300mL tap water and 10 g yeast (at 24% solids). Dough was leavened andbaked using a Breville Bakers Oven on setting 3B (HWI Electrical, SydneyAustralia). The yeast raised (leavened) the dough mixture to produce aloaf of bread with height of 14 cm.

Glucan content. An amount of yeast biomass equivalent to 59 mg drymatter was extracted for glucans according to the method described bySutherland I W, and Wilkinson J F (1971) “Chemical Extraction Methods ofMicrobial Cells”, in Methods in Microbiology Vol 5B (Eds. J R Norris andD W Ribbons), Chapter IV pp. 345-383, Academic Press London and NewYork. According to Sutherland et al. this method produces “cell wallglucan which is free from contaminating material”. Using the describedprocedure an amount of 8 mg of glucan was obtained from the dry yeaststarting material.

Amino Acid/Protein content. An amount of yeast material equivalent to24.6 mg dry yeast matter was suspended in a glass test tube in 2.5 mL of1 M NaOH and placed in a boiling water bath for 15 min. The boiledsample was cooled to 22° C. and volume made to 10 mL using distilledwater. Amino acid/protein was assayed according to Lowry O H, RosebroughN J, Farr A L, and Randall R J (1951) Journal of Biological Chemistry193:265-275, using a bovine serum albumin standard. This assay indicatedthe yeast contained equivalent of 39% amino acid/protein material per wtdry matter.

Nucleotide content. An amount of yeast material equivalent 8.3 mg of dryyeast was resuspended in 1 mL of 4% w/v NaCl and autoclaved at 121° C.for 15 min. Upon cooling to 22° C. the yeast suspension was centrifugedat 3,000×g for 10 min at 22° C. and the supernatant assayed fornucleotide content by the spectrophotometric UV absorbance methoddescribed by Herbert D, Phipps P J, and Strange R E (1971) “ChemicalAnalysis of Microbial Cells”, in Methods in Microbiology Vol 5B (Eds. JR Norris and D W Ribbons), Chapter III pp. 209-344, Academic PressLondon and New York. The yeast contained 1.9% nucleotide material per wtdry matter.

Glutathione content. An amount of yeast material equivalent 6.82 mg ofdry yeast was resuspended in 0.8 mL of 80:20 ethanol:distilled water,vortex mixed and centrifuged at 10,000×g for 2 min at 22° C. Thesupernatant was assayed as follows: Phosphate buffer contained 3.99 gNa₂HPO₄, 0.43 g NaH₂PO₄.H₂O, 0.59 g disodium-EDTA.2H₂O per 250 mL atpH7.5. NADPH solution contained 26.6 mg NADPH tetra-sodium salt in 100mL of phosphate buffer. 5,5″-dithio-bis(2-nitrobenzoic acid) (DTNB) wasprepared by weighing 23.8 mg DTNB in 10 mL of phosphate buffer.Glutathione standard was prepared in distilled water to a stockconcentration of 0.1 mM. Glutathione reductase stock solution fromFluka. Chemie AG contained 162.72 units of activity per mL.

Assay was carried out in a 3 mL spectrophotometer cuvette whichcontained 1.4 mL of NADPH solution+200 microliter DTNB solution+10microliter of sample, standard GSH solution, or distilled water and 390microliter of distilled water. Sample was prewarmed to 30° C. andreaction started by addition of three microliter of glutathionereductase. Cuvettes were incubated at 30° C. for 30 min then theabsorbance read against the distilled water blank at 412 nm wavelengthusing a Shimadzu UV-1201 spectrophotometer. This assay indicated theyeast contained 0.36% total glutathione per wt dry matter.

Phytase activity. An amount of yeast material equivalent to 13.5 mg dryyeast was resuspended in 0.5 mL 0.2M sodium acetate buffer at pH4.9.Phosphatase substrate from Sigma-Aldrich Chemie GmbH was made to 1 mgper mL in 0.2M sodium acetate buffer at pH4.9, and Phytase enzyme fromSigma-Aldrich Chemie GmbH (defined by the supplier as having 1.1 unitsof phytase activity per mg solid material) was made to 0.909 units permL in 0.2M sodium acetate buffer at pH4.9. Assay was performed in acuvette containing 500 microliter of sodium acetate buffer+250microliter of phosphatase substrate solution and 250 microliter ofeither yeast suspension, phytase enzyme or distilled water. Cuvetteswere incubated for 20 min at 30° C. at which time 300 microliter of 10MNaOH was added. Absorbance at 405 nm was read against the distilledwater blank. The phytase activity of the yeast was 0.068 units per mgdry yeast matter.

Invertase activity. An amount of yeast material equivalent to 14.48 mgdry yeast was resuspended in 1 mL of distilled water. The suspension wasfurther diluted one hundred-fold in distilled water. Invertase activitywas assayed according to the colorimetric method described in describedin test T3 of U.S. Pat. No. 4,396,632 and U.S. Pat. No. 5,741,695. Theyeast produced 0.93 units of invertase activity where a unit of activityis defined as 1 micromole of glucose released from sucrose per minute at30° C. and pH4.9 per mg of dry yeast.

These data indicate the potential for non-recombinant Saccharomycescerevisiae to be grown on xylose as the sole carbon source to producebiomass, metabolism, cellular components, and/or enzyme activities thatare relevant to broad types of industrial applications. Those skilled inthe art will know that the exact levels or amounts of these features canbe manipulated not only through classical and recombinant geneticalmethods but also through means of altering culturing conditions, suchthat the results given above are merely indicative.

The invention claimed is:
 1. A method of producing a strain ofSaccharomyces that is capable of growing at a desired growth rate usingxylose as a sole carbon source, the method comprising: (a) providing apopulation of genetically diverse nonrecombinant yeast cells of thegenus Saccharomyces; (b) culturing the yeast cells under conditions thatpermit combining of DNA between the yeast cells; (c) screening orselecting the yeast cells by incubating the yeast cells on or inxylose-containing medium for a time sufficient to permit yeast cellscapable of growing on xylose-containing medium to grow; and (d)isolating one or more of the screened or selected yeast cells having agrowth rate of at least one generation per 48 hours using xylose as asole carbon source, wherein steps (b) and (c) are optionally repeateduntil one or more yeast cells having a growth rate of at least onegeneration per 48 hours can be isolated.
 2. The method of claim 1,wherein the growth rate is at least one generation per 24 hours usingxylose as a sole carbon source.
 3. The method of claim 1, wherein theyeast cells are incubated on xylose-containing medium.
 4. The method ofclaim 1, wherein the xylose-containing medium contains xylose as thesole carbon source.
 5. The method of claim 1, wherein the yeast cellsare selected by incubating the yeast cells on the xylose-containingmedium.
 6. The method of claim 1, wherein the xylose-containing mediumis xylose minimal medium.
 7. The method of claim 1, wherein thexylose-containing medium is solid xylose-containing medium.
 8. Themethod of claim 1, wherein the combining of DNA between the yeast cellsoccurs by mating.
 9. The method of claim 8, wherein the mating occurs bysporulation of the yeast cells and hybridizing of sexually competentoffspring.
 10. The method of claim 1, wherein the population ofgenetically diverse yeast cells comprises the species Saccharomycescerevisiae.
 11. The method of claim 1, wherein the population of yeastcells comprises naturally occurring Saccharomyces strains.
 12. Themethod of claim 10, wherein the xylose-containing medium is one in whichxylose is the sole carbon source.
 13. The method of claim 10, whereinthe combining of DNA between the yeast occurs by mating.
 14. The methodof claim 1, wherein the population of genetically diverse nonrecombinantyeast cells of the genus Saccharomyces includes one or more of: S.cerevisiae, S. paradoxus, S. mikatae, S. cariocanus, S. kudriavzevii, S.pastorianus, and S. bayanus.